JP2005522671A - Assay buffer, composition containing it, and method of use thereof - Google Patents

Abstract

Compositions, reagents, kits, systems, system components and methods for performing assays. More particularly, the present invention relates to the use of novel combinations of reagents to improve assay performance.

Description

  This application claims priority from US Provisional Application No. 60 / 318,289, filed on Sep. 10, 2001, and US Provisional Application No. 60 / 363,498, filed on Mar. 11, 2002. To do. Both of these US provisional applications are incorporated herein by reference.

  This application relates to compositions for use in assays, particularly electrochemiluminescence assays, and methods of use thereof.

  Currently, there are several commercially available instruments that utilize electrochemiluminescence (ECL) for analytical measurements including drug screening. Species that can be induced to release ECL (ECL active species) have been used as ECL labels. Examples of ECL labels include i) organometallic compounds in which the metal is a noble metal of group VIII, including ii) luminol and There are related compounds. Species that participate in the ECL process with ECL labels are referred to herein as ECL coreactants. Commonly used co-reactants include tertiary amines for ECL from RuBpy (see, eg, US Pat. No. 5,846,485, incorporated herein by reference), oxalates and persulfates, As well as hydrogen peroxide to ECL derived from luminol (see, eg, US Pat. No. 5,240,863). The light generated by the ECL label can be used as a reporter signal in diagnostic procedures (Bard et al., US Pat. No. 5,238,808). For example, the ECL label can be covalently linked to a binding agent such as an antibody, nucleic acid probe, receptor or ligand. By measuring the ECL emitted from the ECL label, the involvement of the binding reagent in the binding interaction can be monitored. Alternatively, the chemical environment can be indicated by the ECL signal from the ECL active compound (see, eg, US Pat. No. 5,641,623, which describes an ECL assay that monitors the formation or destruction of ECL co-reactants). Wanna) For more detailed background on ECL, ECL labeling, ECL assays and instrumentation for performing ECL assays, see US Pat. Nos. 5,093,268, 5,147,806, 5,324. No. 457, No. 5,591,581, No. 5,597,910, No. 5,641,623, No. 5,643,713, No. 5,679,519, No. 5,705,402, 5,846,485, 5,866,434, 5,786,141, 5,731,147, 6,066, No. 448, No. 6,136,268, No. 5,776,672, No. 5,308,754, No. 5,240,863, No. 6,207,369, and No. 5,589,136 and International Publication No. 99/633 No. 7, No. 00/03233, No. 99/58962, No. 99/32662, No. 99/14599, No. 98/12539, No. 97/36931 and No. 98/57154 Please refer to the issue.

  Commercially available ECL equipment exhibits exceptional performance. These instruments have become widely used for reasons including excellent sensitivity, dynamic range, accuracy, and complex sample matrix tolerances. Commercial instrumentation uses a flow cell based design that includes a permanently reusable flow cell. Recently, ECL measurement means using reagents used to induce ECL immobilized on electrodes have been disclosed (eg, US Pat. Nos. 6,140,045, 6,066,448, Nos. 6,090,545, 6,207,369 and WO 98/12539). Multiwell plates having such integrated electrodes suitable for such ECL measurements have also been recently disclosed (eg, “Assay Plates for Luminescent Test Measurements, each filed on June 28, 2002, incorporated herein by reference”). US Pat. Application Nos. 10 / 185,274 and 10 / 185,363 entitled “Assay Plates, Reader Systems and Methods for Luminescence Test Measurements”. US Patent Application No. Glezer et al. (Methods and Apparatus for Conducting Measurements, Methods and Apparatus for Conducting Measurements), filed on the same day as this application and incorporated herein by reference. on a Sample) ”[reference number 100405-06410]).

  Currently, pH buffers containing inorganic phosphate are used in a number of electrochemiluminescence assays. Applicants have discovered that such pH buffers may interfere with the assay and reduce the performance of the assay in certain assays.

  Accordingly, there is a need to find an alternative pH assay buffer, a composition containing the pH assay buffer, and methods of use thereof for use in assays that are adversely affected by inorganic phosphate-containing pH buffers. Yes. There is also a need to find alternative ECL assay buffers with improved performance in ECL assays.

  The present invention relates to improved compositions, reagents, kits, systems, system components and methods for performing assays. More particularly, the invention relates to the use of novel combinations of reagents that improve assay performance.

  One aspect of the invention relates to an ECL co-reactant and, preferably, an improved ECL assay buffer comprising a pH buffer. This ECL assay buffer provides a suitable environment for measuring ECL label with high sensitivity by efficiently inducing ECL label to generate ECL and measuring ECL. The ECL assay buffer of the present invention can optionally include additional components including detergents, preservatives, antifoams, ECL active species, salts, metal ions and / or metal chelators. The ECL assay buffer of the present invention can also include bioassay components including binding reagents, enzymes, enzyme substrates, cofactors and / or enzyme inhibitors, and in some cases, labeling bioassay components with ECL labels. can do. The present invention also includes assay reagents, compositions, kits, systems and system components comprising the ECL assay buffer of the present invention and optionally additional assay components. The invention also includes a method of performing an ECL assay using the ECL assay buffer of the invention.

  Another aspect of the invention relates to the use of a pH buffer substantially free of inorganic phosphate. Such buffers have been found to significantly improve ECL measurement performance in some applications. Such buffers have also been found to be advantageous in certain applications where phosphate has been found to interfere with chemical, biochemical or biological reactions.

  Surprisingly, such reagents include several enhancements, including improved performance of assays using phosphate-specific antibodies (ie, antibodies that specifically bind to phosphopeptides, phosphoamino acids and / or phosphoproteins). That amazing advantage. These antibodies probably have a low affinity for inorganic phosphate, and the absence of inorganic phosphate is thought to greatly reduce interference between pH buffer phosphates and phosphate-specific antibodies. Accordingly, the present invention is for measuring phosphopeptides, phosphoamino acids or phosphoproteins using a buffer composition that is free or substantially free (eg, below a level that interferes with phosphate-specific antibodies). Methods, reagents, kits and compositions are included. Such methods, kits, compositions and reagents are preferably applied to the measurement of protein kinase or phosphorylase activity (most preferably using ECL detection) by specific measurement of reaction products or substrates.

  Another aspect of the invention relates to compositions and reagents with high signal / background ratios in electrochemiluminescence assays. Such performance improvements are identified by identifying advantageous combinations of ECL co-reactants, pH buffers, detergents and pH, in particular ECL co-reactants and / or pH buffers other than TPA and phosphate. Achieved through the use of These improved formulations are particularly valuable in non-wash assays and sensitive assays. In some embodiments of the invention, the performance of the ECL assay is further improved by optimally combining the reagent composition and the electrode composition.

  In some embodiments of the present invention, the compositions and reagents of the present invention improve the ratio of ECL signal from bound label to ECL signal from free label. This is especially true for assays involving reagents immobilized on solid surfaces such as electrodes. This is important, for example, in solid phase assays where there is no wash step (especially in low affinity interaction assays). This is because the main part of the background signal comes from the label present in the solution.

  Yet another advantage of the present invention relates to increasing the sensitivity of assays using the compositions of the present invention. More specifically, the ECL assay buffer of the present invention reduces background electrochemiluminescence in the absence of ECL label and improves sensitivity at low detection levels. Surprisingly, an ECL assay buffer comprising a pH buffer other than phosphate or substantially free of inorganic phosphate is a conventional ECL assay buffer comprising an inorganic phosphate-based pH buffer. Less background light emission. This is particularly advantageous at low detection levels where assay performance is greatly improved by increasing the signal / background ratio.

  Another aspect of the invention is that the reagent comprises a non-phosphate based pH buffer, the ECL assay buffer is substantially free of inorganic phosphate, and / or the ECL assay buffer is a second agent other than TPA. It relates to an improved reagent kit comprising an ECL assay buffer, wherein a tertiary amine co-reactant is used. In particular, a kit comprising an ECL assay buffer in one or more containers, preferably also comprising one or more other assay components.

  Another aspect of the present invention relates to improved methods implemented using the present invention, in particular assay methods using phosphate specific antibodies, low detection limits, immobilized reagents and / or non-washed formats.

  Yet another aspect of the present invention relates to an improved system and apparatus comprising the composition or reagent of the present invention, and / or an improved system and apparatus configured to perform the improved method of the present invention.

  The present invention, as well as further objects, features and advantages thereof, will be more fully understood from the following detailed description of certain preferred embodiments.

  The ECL active species can also be called an ECL component, ECL label, ECL label compound, ECL label substance, and the like. Other molecules (especially when used in any of the embodiments of the composition, reagent, kit, method or system according to the invention), in particular components of biochemistry or bioassays, such as analytes or analogs thereof, analytes These ECL active species linked to a reactive component capable of binding to an analyte, an analog thereof, or a binding partner thereof, or a further binding partner of such a binding partner as described above, or an analyte, an analog thereof or a binding partner as described above. Within the scope of the invention. The species described above can also be linked to one or more binding partners and / or a combination of one or more reaction components. In certain enzyme assays, ECL active species can be linked to an enzyme substrate.

  The above-mentioned “composition”, sometimes referred to hereinafter as “ECL, composition” or “system”, is an unstable species formed during the ECL reaction, such as the ECL component in the excited state, and the strong reducing agent, It is within the scope of this invention to contain metastable species, and other intermediate species as well. In addition, although the emission of visible light is an advantageous feature of certain embodiments of the present invention, compositions that emit other types of electromagnetic radiation such as infrared or ultraviolet light, X-rays, microwaves (hereinafter sometimes referred to as Also referred to as an “ECL composition”) or system is within the scope of the present invention. The use of the terms “electrochemiluminescent”, “electrochemiluminescent”, “electrochemiluminescent”, “luminescent”, “luminescent” and “luminescent” in relation to the present invention means that emission is light The emission may be in such other forms of electromagnetic radiation.

The present invention relates to ECL assay buffers, assay compositions containing them, and methods of use thereof. As mentioned above, several disadvantages were discovered when using prior art phosphate-based ECL assay buffers. More specifically, phosphate-specific antibodies by phosphate in a standard formulation of ECL co-reactant TPA (ORIGEN assay buffer, IGEN International: 200 mM phosphate, about 100 mM TPA, pH about 7.5) It was found during the development of an ECL assay specific for tyrosine kinase activity that the binding between and phosphorylated substrates is broken. Assays include i) kinase-dependent phosphorylation of peptides immobilized on a carbon electrode, ii) specific binding of labeled phosphate-specific antibodies (by Ru (bpy) ₃ derivatives), iii) ECL co-reaction Addition of tripropylamine (TPA), and iv) detection of ECL from the bound label (see, eg, Example A below). Applicants have shown that when TPA is added by the addition of ORIGEN assay buffer, the measured ECL signal is clearly dependent on the time the binding complex has been exposed to the ORIGEN assay buffer (Figure 1). We found that the signal dropped sharply with time. In fact, only a small portion (about 10%) of the initial signal was detected after 1 hour incubation. The affinity of the pY20 antibody (Zymed Lab) used in the assay for the phosphotyrosine sites formed on the plate surface during the enzymatic reaction was much greater than the affinity for free phosphate in solution. However, it is currently believed that due to the high free phosphate concentration in the ORIGEN assay buffer (200 mM), the phosphotyrosine / pY20 complex dissociates, resulting in a rapid decay of the signal.

  One way to circumvent this problem is to fix the time from administration of the ECL assay buffer to the reading step and to calibrate and subtract the signal decay. However, this approach is not desirable in high throughput screening applications where assay robustness and administration protocol flexibility are desired.

  Thus, several different organic pH buffering agents have been tested as replacements for traditional phosphate-based assay buffers. However, many of the conventional biological buffers (including Tricine, all made by Sigma, HEPES, MOPS, BES) interfere with ECL generation from TPA and are observed with ORIGEN assay buffers. It gave only 2-20% of the signal. However, Applicants have discovered a set of buffers that give an ECL signal comparable to that observed in TPA / phosphate.

  Therefore, Applicants have replaced the phosphate buffer with a pH buffer that is substantially free of inorganic phosphate, eliminating the above-mentioned drawbacks and equivalent to the signal observed in standard ORIGEN assay buffer. It was discovered that ECL signal can be generated. It is preferable that the pH buffer does not contain an inorganic buffer.

  Applicants can also not only benefit the phosphate-free ECL assay buffer of the present invention when applied to phosphopeptide binding assays, but also improve a wide range of ECL assays. It has been found that it also has other advantageous properties (including a low ground signal).

  Applicants have also discovered an ECL assay buffer background reducing agent that, when introduced into an ECL assay buffer, reduces the background of the ECL assay buffer and improves assay performance. These reducing agents are also preferably pH buffering agents, most preferably GlyGly or Tris.

  Applicants have also discovered a novel ECL assay buffer that uses ECL co-reactants other than conventional TPA. Surprisingly, it has been discovered that some co-reactants generate an ECL signal comparable to that generated by TPA. In addition, since ECL co-reactants other than TPA have improved performance over TPA, the use of ECL co-reactant improves the non-washing ECL assay performance, and it is released into the ECL label held in the vicinity of the electrode and in solution. It was found that the markings were identified. The use of a co-reactant other than TPA has another advantage due to the high water solubility and low vapor pressure of some of the identified new co-reactants.

  Applicants have also discovered that the presence or absence of detergents can have a significant impact on the performance of ECL assay buffers. Surprisingly, the effectiveness of the cleaning agent on the ECL can depend on what is chosen for the ECL co-reactant and the working electrode material. Applicants have developed detergent-containing ECL assay buffers suitable for a variety of different applications and ECL systems.

  As mentioned above, one aspect of the present invention relates to an improved ECL assay buffer comprising an ECL co-reactant and preferably a pH buffer. This ECL assay buffer provides a suitable environment for measuring ECL label with high sensitivity by efficiently inducing ECL label to generate ECL and measuring ECL. The ECL assay buffer of the present invention can optionally include additional components including detergents, preservatives, antifoams, ECL active species, salts, metal ions and / or metal chelators. The ECL assay buffer of the present invention can also include bioassay components including binding reagents, enzymes, enzyme substrates, cofactors and / or enzyme inhibitors, and in some cases, labeling bioassay components with ECL labels. can do.

  Preferably, the ECL assay buffer of the present invention is essentially aqueous or substantially aqueous, although there are applications where it is desirable to add organic cosolvents such as DMSO, DMF, methanol, ethanol, other alcohols. is there. In one embodiment of the invention, the ECL assay buffer (or one or more components thereof) is provided in a dried form, and the user can provide an appropriate solvent or matrix (preferably water or an aqueous medium). Make an ECL assay buffer solution by adding.

5.1 ECL Co-reactant Most, if not all, applications of current commercial ECL technology include tri-n-propylamine (TPA) as a co-reactant and phosphate as a pH buffer. Measurement of ECL label (and in particular, organometallic complexes of ruthenium) in the presence of the containing ECL assay buffer is included. These ECL assay buffers are optimized for commercial ECL instrumentation that uses magnetic particles collected on a metal (generally platinum) electrode surface as a solid phase for binding assays and exhibits superior performance.

Applicants have discovered that in some applications, tertiary alkyl amines with certain functionalities can perform as well or better than TPA. These functional tertiary amines can be used for carbon-based electrodes (for example, electrodes including composites including carbon particles or carbon nanotubes such as plastics, inks) and / or assay reagents (binding reagents) immobilized on the electrodes. Etc.) are particularly useful in assays using. The functional tertiary alkyl amines of the present invention preferably have one or more of the following properties. i) Functional tertiary alkyl amines can be oxidized on a carbon-based electrode with one electrode oxidation to amine radical cations, which can subsequently lose protons to form radical reducing agents. (Scheme 1). ii) On the carbon base electrode, it has an oxidation potential equivalent to (within 150 mV) or higher than that of Ru (II) (bpy) ₃ . iii) Most preferably at pH 6-9, it can be oxidized at a carbon based electrode at a potential lower than that required to decompose water. energy iv) a radical reducing agent and Ru (III) (bpy) 3 is released by the reaction which produces Ru (II) _{(bpy) 3} by reaction, Ru luminescent excited state (II) the _{(bpy) 3} Enough to produce. v) The lifetime of the amine radical cation and / or radical reducing agent is shorter than the corresponding TPA-derived species.

  Applicants have used the functional tertiary alkylamines of the present invention for the ECL excitation at the electrode relative to the ECL label bound to the electrode (as opposed to the ECL label free in solution). It was discovered that it is possible to improve selectivity. Without being bound by theory, this increased selectivity is due to the fact that the lifetime of the amine radical cation and / or radical reducing agent is shorter than the corresponding TPA-derived species (and thus the response to the ECL reaction occurring near the electrode surface). This is thought to be because the involvement of sex species is limited. Preferably, the diffusion distance of the amine radical cation and / or radical reducing agent (the distance that the species can diffuse during its lifetime) is less than 1 μm, more preferably <500 nm, even more preferably less than 100 nm, More preferably less than 50 nm, most preferably <10 nm. The high selectivity between free and bound labels improved sensitivity in the non-washed ECL assay format. The signal ratio (B / F ratio) from the bound label to the free label can be improved by replacing TPA with the non-TPA co-reactant of the present invention. This improvement is preferably more than 2 times, more preferably more than 5 times, and most preferably more than 10 times.

The functional tertiary amine co-reactant of the present invention preferably has a structure of NR ¹ R ² R ³ . Wherein R ¹ , R ² and R ³ are alkyl groups containing at least 2, preferably 3 carbons, and one or more of R ¹ , R ² and R ³ are more preferably hydrophilic functional groups. Is functionalized with a charged group, most preferably a negatively charged group. Preferred functional groups include hydroxyl, dialkylamino, sulfate, sulfonate, carboxylate, carboxylic acid ester and the like.

Particularly preferred co-reactants include compounds having a (n-Pr) ₂ N (CH ₂ ) _n R structure. Where n is 2 or more (more preferably 3) and R is a hydrophilic functional group as defined above, preferably carboxylate, dialkylamino (more preferably dipropylamino), most preferably sulfonate. .

  Other preferred co-reactants include compounds having the following structure.

Wherein i) X is — (CH ₂ ) — or a heteroatom, preferably —O—, —S—, or —N (R ¹ ) —, and ii) R and R ¹ are two or more ( Preferably 3 or more) carbon-containing alkyl groups, iii) n and m are each 1 or more, preferably 2, iv) R (and optionally R ¹ ) is hydrophilic as defined above Contains functional groups. Most preferably, R is — (CH ₂ ) _n —F ¹ . In the formula, n is 3 or more, and F ¹ is a hydrophilic functional group, preferably carboxylate or sulfonate. When X is —N (R ¹ ) —, R ¹ is most preferably — (CH ₂ ) _n —F ² . Where n is 3 or more and F ¹ is H, alkyl or a hydrophilic functional group, most preferably carboxylate or sulfonate.

  The so-called “Good” buffer (Good et al., Biochemistry, 5, 467 (1966); Good et al., Methods in Enzymol., 24, Part B, 53 (1972), and Ferguson et al., Anal. Biochem., 104, 300 ( Many of 1980)) were found to contain tertiary amines and act as ECL co-reactants on the carbon electrode. These “good” buffering agents generally include tertiary amines having a piperazine or morpholine nucleus. Specific amines that act as ECL coreactants on the carbon-based electrode include 3- (di-n-propylamino) -propanesulfonic acid, 4- (di-n-propylamino) -butanesulfonic acid, 4- [Bis- (2-hydroxyethane) -amino] -butanesulfonic acid, piperidine-N- (3-propanesulfonic acid), azepane-N- (3-propanesulfonic acid), piperidine-N- (3-propionic acid ) (PPA), 3-morpholino-2-hydroxypropanesulfonic acid (MOPSO), 3-morpholinepropanesulfonic acid (MOPS), N- (2-hydroxyethyl) piperazine-N′-3-propanesulfonic acid (EPPS) N- (2-hydroxyethyl) piperazine-N′-3-ethanesulfonic acid (BES), piperazine-N, N′-bi (2-ethanesulfonic acid) (PIPES), triethanolamine, N-2-hydroxypiperazine-N-2-ethanesulfonic acid (HEPES), piperazine-N, N′-bis-4-butanesulfonic acid, homopiperidine -N-3-propanesulfonic acid, piperazine-N, N'-bis-3-propanesulfonic acid, piperidine-N-3-propanesulfonic acid, piperazine-N-2-hydroxyethane-N'-3-methylprop Noate, piperazine-N, N′-bis-3-methylpropanoate, 1,6-diaminohexane-N, N, N ′, N′-tetraacetic acid, N, N-bispropyl-N-4 -Aminobutanesulfonic acid, N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (TES), 1,3-bis [tris (hydroxymethyl) Methylamino] propane (bis-Trispropane), 3-dimethylamino-1-propanol, 3-dimethylamino-2-propanol, N, N, N ′, N′-tetrapropylpropane-1,3-diamine (TPA2) ), Piperazine-N, N′-bis (2-hydroxypropane) sulfonic acid (POPSO), 2-hydroxy-3- [4- (2-hydroxyethyl) piperazin-1-yl] propane-1-sulfone Acid (HEPPSO) and the like. HEPES, POPSO, HEPPSO, EPPSO, PPA, and PIPES are particularly preferable because of high signal and easy identification of bound label and free label. TES is also preferred because of its high signal.

  Additional co-reactants include proline, peptides having an N-terminal proline, and the like. Proline is preferably N-alkylated to form a tertiary amine.

  The use of co-reactants with hydrophilic functional groups (and in particular co-reactants that are zwitterions at neutral pH) has various advantages regardless of their ability to act as ECL co-reactants. These species tend to have high water solubility and low vapor pressure. Therefore, it is possible to produce highly concentrated stock solutions that are diluted as needed during use. There is no instability of disappearance of the co-reactant into the gas phase, and it is possible to prepare a dry reagent containing the co-reactant. Also, these co-reactants dissolve quickly to a minimum volume when they are dry reagents.

5.2 pH Buffers Conventional ECL assay buffers optimized for use with commercial ECL instruments generally include TPA in phosphate-based pH buffers. These formulations were particularly useful for performing solid phase assays using magnetic particles captured on electrodes. Applicants have discovered that in some applications, other pH buffers (including organic pH buffers) can provide performance beyond phosphate. These non-phosphate pH buffering agents (and pH buffering solutions, and ECL assay buffers containing these buffering agents, are based on carbon-based electrodes (e.g. carbon particles such as plastics and inks or carbon nanotube-containing composites). Electrode) and / or assays using assay reagents immobilized on the electrode (such as binding reagents), even when used in assays where phosphate is an interferent. Preferably, an ECL assay buffer using a non-phosphate buffer of the invention is less than 15 mM inorganic phosphate, more preferably less than 5 mM inorganic phosphate, even more preferably less than 1 mM. Contains phosphate, even more preferably substantially free of inorganic phosphate, most preferably contains inorganic phosphate There.

  The pH buffer is preferably not oxidized under the conditions used to release ECL and does not interfere with the production of ECL. Two pH buffering agents that have proven particularly useful are tris (hydroxymethyl) aminomethane (Tris) and oligo (glycine), preferably glycyl-glycine (Gly-Gly). Applicants have reported that in ECL assays on a carbon-based electrode using TPA / Tris or TPA / Gly-Gly ECL assay buffer, the signal from the ECL label bound to the electrode is a conventional TPA / phosphate buffer. It was found that the signal is equivalent to that observed by the agent. However, the background signal in the absence of ECL label is much less with Tris and Gly-Gly buffers. The decreased background signal resulted in a higher signal / background ratio (S / B) and increased sensitivity of the ECL assay with the new formulation. The S / B ratio of the ECL assay buffer of the present invention is preferably 2 times, more preferably 5 times, most preferably 10 times better than the S / B ratio obtained using a phosphate-based system. .

  Without being bound by theory, applicants have found that the performance enhancement of Tris and Gly-gly based ECL assay buffers is responsible for tertiary amine oxidation products and / or others that are responsible for the assay buffer background. It is hypothesized to be related to the ability of the buffer to act as an ECL assay buffer reducing agent by reacting with and destroying reactive oxidizing species (eg, amine radical cations and radical reducing agents) . This effect is most noticeable where the concentration of these species is lower away from the electrode surface and therefore the Tris and Gly-gly components do not affect the signal from the electrode-bound label. Tris and Gly-gly buffers can also improve the observed bound to free ratio. However, this effect is less than that observed by substituting a non-TPA buffer such as PIPES.

  Applicants have found that the Tris and Gly-gly buffer systems are also suitable for use with non-TPA co-reactants such as PIPES. If a co-reactant such as PIPES, which can act as a pH buffer, is used, additional buffer can be omitted.

5.3 Detergent Applicants have discovered that the presence or absence of a detersive agent can have a surprisingly large effect on the ECL signal. The nature of this effect is unexpectedly determined by the electrode. On oxidized electrodes (eg, plasma oxidized carbon inks or plasma oxidized polymer composites containing carbon particles or carbon nanotubes) exposed to a TPA-containing ECL assay buffer, this effect is demonstrated by Triton, Nonidet series detergents (eg, It appears to be relatively small except in the case of phenyl ether containing detergents such as Triton X-100). A common way to generate ECL is to use a ramp potential. In general, the plot of ECL intensity and applied potential has a peak shape. ECL increases as the oxidation potential of the label approaches that of the co-reactant. If scanning continues past this potential, the co-reactant is eventually consumed, the ECL intensity begins to decrease, and water oxidation begins to occur. Applicants have observed that adding a phenyl ether-containing detergent adds a small ECL peak at a higher potential than the ECL main peak. This peak is at approximately the same potential as the oxidation wave observed with pure Triton X-100, so Applicants have found that the new peak is the oxidation of the detergent (or accompanying impurities) and its effect on the ECL reaction. Presumed to be related to the involvement of oxidation products.

  The behavior with non-oxidized carbon-based electrodes (and especially untreated carbon ink electrodes) is very different. For these electrodes, the ECL signal (as well as the S / B ratio) in the presence of TPA-containing buffer was greatly improved by the addition of detergent. Although this effect seems to be relatively independent of the nature of the detergent (nonionic detergents are preferred because of their relatively weak ability to denature biological systems), the detergent concentration is the critical micelle concentration of the detergent. It is necessary to be approximately equal to or greater than (cmc). In a preferred embodiment of the invention, by adding a detergent to the ECL assay buffer, the assay signal or S / B (preferably induced by a carbon-based electrode, most preferably a carbon ink electrode) is 2 The improvement is more than twice, more preferably more than 5 times, and most preferably more than 10 times.

  Non-TPA containing ECL assay buffer, especially non-TPA containing ECL assay buffer (especially a buffer comprising a non-TPA tertiary amine co-reactant of the invention, preferably N-substituted morpholine or piperazine, most preferably PIPES ) Appears to be less dependent on the nature of the carbon electrode. For example, Applicants have shown that assays using PIPES as a co-reactant can be unexpectedly improved by adding phenyl ether containing materials, particularly phenyl ether containing detergents, both on oxidized and non-oxidized electrodes. I found. Other detergents that did not contain a phenyl ether component did not show this effect. In a preferred embodiment of the invention, when a detergent is added to a non-TPA based ECL assay buffer (preferably a PIPES based ECL assay buffer) (preferably a carbon based electrode, most preferably a carbon ink electrode). The assay signal or S / B induced) is improved by more than 10 times, more preferably more than 30 times, most preferably more than 100 times.

In certain assays, eg, assays involving detergent sensitive components such as biological membranes, it may be advantageous to reduce (eg, to <0.1%) or eliminate detergent from the ECL assay buffer. . It should be understood that the various detergent-containing formulations of the present invention can be prepared for these detergent-sensitive applications in a cleaner-less form or a detergent-free form. In a preferred embodiment, in assays using detergent-sensitive components, the following co-reactants are TPA, N, N-bis- (hydroxyethyl) -N-4-aminobutanesulfonic acid or A ₂ N— (CH ₂₎ ECL assay buffer containing one of the _n -NB ₂ is used. In the formula, A and B are alkyl groups (preferably propyl), and n is an integer (preferably 3 or 4, most preferably 3).

5.4 Preservatives When storing ECL assay buffers, it may be advantageous to include preservatives that prevent microbial growth. The preservative preferably has little or no effect on the ECL generated with the ECL assay buffer, especially when using the ECL assay buffer with a carbon-based electrode. Azide has been found to be a suitable preservative. Isothiazolones (eg Kathon, 2-methyl-4-isothiazolin-3-one and 5-chloro-2-methyl-4-isothiazolin-3-one), oxazolidine (eg Oxaban A or 4,4 dimethyloxazolidine) and relationships Such preservatives are particularly preferred because of their high compatibility with ECL, high activity, fewer problems associated with safety issues or environmental issues.

5.5 Antifoam agents It may be advantageous to avoid the formation of bubbles, ie bubbles, especially in HTS applications. For this reason, it may be desirable to add an antifoam agent to the ECL assay buffer. Applicants have found that a number of commercially available antifoams (including Antifoms o-30, Antifoam 204, Antifoam A, Antifoam SE-15, Antifoam SO-25 and Antifoam 289) are significant in the performance of ECL assay buffers. It was found that it can be added to the ECL assay buffer without adverse effects.

5.6 ECL Label The composition of the present invention can comprise an ECL label. The ECL label may be a conventional ECL label. Examples of ECL labels include i) organometallic compounds in which the metal is selected from, for example, Group VIII noble metals, including Ru-containing and Os-containing organometallic compounds such as a tris-bipyridyl-ruthenium (RuBpy) component, and ii ) Luminol and related compounds. The ECL label is preferably capable of repeatedly releasing electrochemiluminescence. Preferred ECL labels are ruthenium or osmium containing organometallic species. More preferably, these ruthenium or osmium containing organometallics comprise ruthenium or osmium chelating with a polypyridyl ligand (most preferably bipyridine, phenanthroline and / or substituted derivatives thereof). Most preferably, the ECL label comprises ruthenium-tris-bipyridine and the bipyridine ligand is optionally substituted, for example with a linking group that couples the label to the assay reagent.

  The ECL label may be linked to the assay reagent, optionally by a linking group. Examples of binding reagents that can be linked to ECL labels include whole cells, cell surface antigens, subcellular particles (eg, organelles or membrane fragments), viruses, prions, dust mites or fragments thereof, viroids, antibodies, Antigen, hapten, fatty acid, nucleic acid (and synthetic analog), protein (and synthetic analog), lipoprotein, polysaccharide, inhibitor, cofactor, hapten, cell receptor, receptor ligand, lipopolysaccharide, glycoprotein, peptide, poly Peptide, enzyme, enzyme substrate, enzyme product, second messenger, cellular metabolite, hormone, drug, synthetic organic molecule, organometallic molecule, tranquilizer, barbiturate, alkaloid, steroid, vitamin, amino acid, sugar, lectin, pair Replacement or derived protein, biotin, avidin, streptavidin . The assay reagent is preferably useful as a binding reagent or enzyme substrate, for example, in a binding assay or enzyme assay.

5.7 Compositions One aspect of the invention pertains to compositions comprising an ECL assay buffer of the invention.

  Another aspect of the invention relates to a composition suitable for use in an assay comprising a pH buffer substantially free of inorganic phosphate. Suitable pH buffering agents include glycylglycine (“Glygly”), tris (hydroxymethyl) aminomethane (“Tris”) or combinations thereof. Other pH buffering agents that are also substantially free or completely free of inorganic phosphate are also suitable for use in the present invention.

According to one embodiment of the invention, the composition comprises a pH buffer, preferably substantially free of inorganic phosphate, preferably one or more ECL co-reactants (preferably TPA. Or a non-TPA co-reactant, more preferably N-substituted morpholine or piperazine, most preferably PIPES). According to a particularly preferred embodiment, the composition does not contain inorganic phosphate. Suitable pH buffering agents include glygly, tris and the like. Further buffering agents have certain preferred properties, i) buffering capacity at pH 6.5-8.5, preferably 7-8 (more preferably, the pKa of the buffer is 6.5-8.5, more preferably Is in the range of 7.5 to 8.5), ii) low commercial availability, iii) no inhibitory effect on ECL, and / or iv) 0 to 1.2V, more preferably 0 The selection can be based on the absence of significant oxidation waves in the range of ~ 1.5V (voltage window for oxidation of Ru (bpy) ₃ and TPA).

According to another embodiment of the present invention, the composition comprises a non-phosphate pH buffer, preferably one or more ECL co-reactants (preferably TPA or non-TPA co-reactants, and more Preferably N-substituted morpholine or piperazine, most preferably PIPES). Preferably, the composition comprises less than 15 mM inorganic phosphate, more preferably less than 5 mM inorganic phosphate, even more preferably less than 1 mM phosphate, even more preferably substantially free of inorganic phosphate. And most preferably free of inorganic phosphate. Suitable pH buffering agents include glygly, tris and the like. Further buffering agents have certain preferred properties, i) buffering capacity at pH 6.5-8.5, preferably 7-8 (more preferably, the pKa of the buffer is 6.5-8.5, more preferably Is in the range of 7.5 to 8.5), ii) low commercial availability, iii) no inhibitory effect on ECL, and / or iv) 0 to 1.2V, more preferably 0 The selection can be based on the absence of significant oxidation waves in the range of ~ 1.5V (voltage window for oxidation of Ru (bpy) ₃ and TPA).

  The ECL co-reactant used in these embodiments is preferably suitable for use in an electrode induced luminescence reaction (eg, electrochemiluminescence). Suitable ECL co-reactants include tripropylamine (TPA). Non-TPA co-reactants (preferably tertiary amines other than TPA as described in the co-reactants section above) are advantageous in some applications, particularly in non-washing format assays.

  The composition preferably comprises a pH buffer of 10 to 2000 mM, more preferably 50 to 1200 mM, even more preferably 100 to 600 mM, and most preferably 300 to 500 mM.

  The composition preferably comprises 10 to 1000 mM, more preferably 30 to 600 mM, even more preferably 50 to 200 mM, most preferably 75 to 150 mM ECL co-reactant.

  The optimal range of TPA concentration in a pH buffer containing Tris and Gly-Gly is very similar to the concentration of ORIGEN® buffer (ie, in the range of 50-200 mM). The test range for Tris and Gly-Gly buffer concentrations is 100-600 mM. Preferably the concentration is 200 mM. An ECL assay buffer containing a non-TPA co-reactant (preferably PIPES) of the present invention can contain approximately the same range of concentrations of co-reactant. However, for many applications, the preferred range is 10-100 mM, most preferably 20-50 mM.

  According to another preferred embodiment, the final formulation of Gly-Gly / TPA buffer is 200 mM Gly-Gly, 100 mM TPA, 0.1% Triton, pH = 7.8 ± 0.05.

  According to another preferred embodiment, the final formulation of Gly-Gly / TPA buffer is 50-1000 mM Gly-Gly, 50-1000 mM TPA, pH = 7.8 ± 1. This formulation preferably also includes 0.2% to 2% Triton X-100 and / or 20 to 500 mM salt.

  According to another preferred embodiment, the final formulation of Tris / TPA buffer is 200 mM Tris, 100 mM TPA, 0.1% Triton, pH = 7.8 ± 0.05.

  According to another preferred embodiment, the final Tris / TPA buffer formulation is 50-1000 mM Tris, 50-1000 mM TPA, pH = 7.8 ± 0.05. This formulation preferably also includes 0.2% to 2% Triton X-100 and / or 20 to 500 mM salt.

  According to another preferred embodiment, the final formulation of PIPES / Phos buffer is 40-1000 mM phosphate (preferably potassium phosphate), 10-200 mM PIPES, pH = 7.8 ± 0.05. . This formulation preferably also includes 0.2% to 2% Triton X-100.

  The use of Tris and Gly-Gly assay buffer significantly improved the stability of the phosphopeptide-antiphosphopeptide complex in ECL-based tyrosine kinase assays. However, it was observed that this complex was partially dissociated in the Tris buffer. However, the rate was much slower than in the ORIGEN assay buffer. In the case of Gly-Gly buffer, the ECL signal gradually increased because no stop reagent to suppress the enzyme reaction was introduced into the assay solution.

According to one preferred embodiment, the composition further comprises a stop reagent (ie, a reagent added to stop the reaction or a reagent added to reduce interference with the reaction). Chelating agents such as ethylenediaminetetraacetic acid (EDTA) are common stop reagents in Mg-dependent kinase assays. EDTA binds to the Mg ²⁺ ions necessary for normal activation of ATP. For example, adding 5 mM EDTA to a Gly-Gly assay buffer helps to stop residual tyrosine kinase enzyme activity. The dissociation of the phosphopeptide-antiphosphopeptide complex in Gly-Gly / TPA buffer containing 5 mM EDTA did not exceed 1% per hour in the non-washed format assay. EDTA can have a negative effect on the absolute value of the ECL signal at concentrations higher than 10 mM, but incubation in assay buffer does not impair the stability of the ECL signal. Depending on the desired final read volume (100 μl or 250 μl) of the 96-well plate and the type of assay (washed or non-washed), the Gly-Gly / TPA solution formulation may vary.

  Preferably, the pH of the composition is 6-9, more preferably 7-8, even more preferably 7.5-8, and most preferably about 7.8. According to one preferred embodiment, the pH is adjusted by adding an acid or base, preferably KOH, more preferably 10% KOH.

One embodiment of the present invention
(A) preferably 0.1-0.7M, more preferably 0.3-0.5M, most preferably about 0.2M glycylglycine (Gly-Gly);
(B) An ECL assay buffer comprising preferably 0.01 M to 0.3 M, more preferably 0.05 to 0.2, and most preferably about 0.1 M tripropylamine (TPA).

  The assay buffer preferably further comprises EDTA (preferably 1-10 mM, more preferably 5 mM). The pH of the assay buffer is preferably 6-9, more preferably 7-8, even more preferably 7.5-8, and most preferably about 7.8. According to one preferred embodiment, the pH is adjusted by adding an acid or base, preferably KOH, more preferably 10% KOH.

Another preferred embodiment of the present invention is:
(A) preferably 0.1 to 0.7 M, more preferably 0.3 to 0.5 M, most preferably about 0.2 M tris (hydroxymethyl) aminomethane (Tris);
(B) An ECL assay buffer comprising preferably 0.01 M to 0.3 M, more preferably 0.05 to 0.2 M, and most preferably about 0.1 M tripropylamine (TPA).

  The assay buffer preferably further comprises 1-10 mM, more preferably 5 mM ethylenediaminetetraacetic acid (EDTA). The pH of the composition is preferably 6-9, more preferably 7-8, even more preferably 7.5-8, and most preferably about 7.8. According to one preferred embodiment, the pH is adjusted by adding an acid or base, preferably KOH, more preferably 10% KOH.

  Another preferred embodiment of the present invention relates to an ECL assay buffer comprising a co-reactant other than TPA, preferably a trialkylamine having a hydrophilic functional group (as described in the co-reactant section). Preferably the co-reactant is PPA or PIPES, most preferably PIPES. The concentration of the co-reactant is preferably 10 to 800 mM, most preferably 10 to 200 mM, and most preferably 20 to 50 mM. The ECL assay buffer preferably also includes a pH buffer, preferably phosphate, Tris or Gly-Gly. The concentration of the pH buffer is preferably 0-800 mM, more preferably 0-400 mM, even more preferably 20-200 mM, most preferably 75-150 mM. The pH of the composition is preferably 6-9, more preferably 7-8, even more preferably 7.2-7.8, and most preferably about 7.5. The ECL assay buffer preferably also includes a substance comprising a phenyl ether component and / or a detergent, preferably a non-ionic detergent, even more preferably a phenyl ether-containing detergent, most preferably Triton X-100. The detergent concentration is preferably higher than 0.02%, more preferably higher than 0.05%, and most preferably higher than 0.05-0.5%.

  According to one preferred embodiment, the reagent or composition of the present invention further comprises one or more detergents and / or surfactants (eg Nonidet, Brij, Triton, Tween, Thesit, Lubrol, Genapol, Pluronic, A class of nonionic detergents / surfactants known under the trade names Tetronic, F108 and Span). Particularly preferred cleaning agents include Tween 20, Triton X-100, NP-40, Thesit and the like.

  Another preferred embodiment of the invention relates to a reagent composition comprising the above-described assay buffer concentrated. The reagent composition is preferably diluted with an aqueous solution, preferably to an assay buffer having an optimal component concentration for use in an assay, preferably an ECL assay.

  Another embodiment relates to a dry reagent precursor that includes one of the above-described dried assay buffers. This dry reagent precursor can preferably be mixed with a solution, preferably an aqueous solution, into an assay buffer solution having an optimal component concentration for use in an assay, preferably an ECL assay.

  Another aspect of the invention is suitable for use in preparing a composition for performing an assay, preferably a luminescence assay, more preferably a chemiluminescence assay, or an electrode induced luminescence assay, most preferably an electrochemiluminescence assay. In addition, the present invention relates to a reagent containing one or more pH buffering agents substantially free of inorganic phosphate.

  Another aspect of the invention is suitable for use in preparing a composition for performing an assay, preferably a luminescence assay, more preferably a chemiluminescence assay or an electrode induced luminescence assay, most preferably an electrochemiluminescence assay. It relates to a reagent containing one or more ECL assay background reducing agents, preferably non-phosphate pH buffering agents. Preferably, the reagent comprises less than 15 mM inorganic phosphate, more preferably less than 5 mM inorganic phosphate, even more preferably less than 1 mM phosphate, and even more preferably substantially comprises inorganic phosphate. And most preferably free of inorganic phosphate.

According to one embodiment, the reagent comprises an ECL assay buffer reducing agent (preferably a non-phosphate pH buffer) and / or is substantially free of inorganic phosphate and performs an ECL assay. Suitable for use in the preparation of compositions for Where electromagnetic radiation is emitted from the assay composition,
(I) a metal-containing ECL component that is convertible from electromagnetic radiation to an excited state;
(Ii) an ECL coreactant (preferably an amine or amine component, most preferably a tertiary amine, most preferably TPA) that forms a strong reducing agent when oxidized;
(Iii) an electrolyte capable of functioning as a medium in which the ECL component and the ECL co-reactant can be oxidized;
A component selected from the group consisting of:

  The reagent preferably comprises the pH buffer, the ECL co-reactant, and one of the other two components of the groups (i) to (iii).

  Another aspect of the invention relates to an assay composition comprising one or more binding reagents, enzymes and / or substrates, and a pH buffering agent of the invention.

  Another aspect of the invention relates to compositions, reagents, kits and methods for performing protein kinase and phosphorylase assays and / or for measuring phosphopeptides, phosphoproteins and phosphoamino acids. One embodiment of the invention relates to a composition comprising a pH buffer and a phosphopeptide-specific antibody and substantially free of inorganic phosphate. It is preferable that the composition does not contain inorganic phosphate. Preferably, the composition further comprises a phosphopeptide, phosphoamino acid and / or phosphilated protein that binds to a phosphopeptide specific antibody.

  Preferably, the pH buffer is selected from the group consisting of glycylglycine, tris (hydroxymethyl) aminomethane, or combinations thereof.

  Preferably, the composition further comprises one or more components selected from the group consisting of kinases and kinase substrates. According to another embodiment, the composition comprises one or more components selected from the group consisting of phosphatases and phosphatase substrates.

  The pH of the composition is preferably 6-9, preferably 7-8, more preferably 7.5-8, and most preferably about 7.8.

  According to one preferred embodiment, the composition further comprises one or more ECL coreactants. The ECL co-reactant preferably contains an amine or an amine component. More preferably, the ECL co-reactant comprises tripropylamine (TPA).

  According to another preferred embodiment, the composition further comprises a stop reagent. The stop reagent preferably includes ethylenediaminetetraacetic acid (EDTA).

  According to another preferred embodiment, the composition further comprises an acid or base, preferably KOH.

  According to one preferred embodiment, the reagent or composition of the present invention further comprises one or more detergents and / or surfactants (eg, Brij, Triton, Tween, Thesit, Lubrol, Genapol, Pluronic, Tetronic, F108 and the class of non-ionic detergents / surfactants known under the Span trade name).

  The composition preferably contains an inhibitor and / or enzyme, more preferably a phosphorylation or dephosphorylation inhibitor and / or enzyme.

  Another embodiment of the invention relates to a composition comprising a pH buffer and an ECL co-reactant and substantially free of inorganic phosphate. It is preferable that the composition does not contain inorganic phosphate.

  The pH buffer is preferably selected from the group consisting of glycylglycine, tris (hydroxymethyl) aminomethane, or combinations thereof.

  Preferably, the composition comprises one or more components selected from the group consisting of kinases and kinase substrates, or one or more components selected from the group consisting of phosphatases and phosphatase substrates.

  The pH of the composition is preferably 6-9, preferably 7-8, more preferably 7.5-8, and most preferably about 7.8.

  The ECL coreactant preferably includes an amine or an amine component. More preferably, the ECL co-reactant comprises tripropylamine (TPA).

  According to another preferred embodiment, the composition further comprises a stop reagent. The stop reagent preferably includes ethylenediaminetetraacetic acid (EDTA).

  According to another preferred embodiment, the composition further comprises an acid or base, preferably KOH.

  The composition preferably contains an inhibitor and / or enzyme, more preferably a phosphorylation or dephosphorylation inhibitor and / or enzyme.

  According to one preferred embodiment, the pH buffer is glycylglycine and the composition further comprises ethylenediaminetetraacetic acid (EDTA) and TPA. It is preferred that the composition further comprises a KOH and / or ECL component.

Another embodiment of the invention relates to a reagent comprising a pH buffer. The pH buffer is substantially free of inorganic phosphate and the reagent is suitable for use in preparing a composition for performing an ECL assay. Where electromagnetic radiation is
(I) a metal-containing ECL component that is convertible to an excited state from which electromagnetic radiation is emitted;
(Ii) an ECL coreactant that forms a strong reducing agent when oxidized;
(Iii) released by an assay composition comprising a component selected from the group consisting of the ECL component and the amine or an electrolyte in which the amine component can function as a medium in which it can be oxidized.

  Preferably, the reagent further comprises the pH buffer, an ECL co-reactant (preferably an amine or an amine component) and one of the other two components of the groups (i) to (iii).

Another embodiment of the invention relates to a reagent comprising a pH buffer. The pH buffer is substantially free of inorganic phosphate and the reagent is suitable for use in preparing a composition for performing an ECL assay. Where electromagnetic radiation is
(I) a metal-containing ECL component that is convertible to an excited state from which electromagnetic radiation is emitted;
(Ii) an ECL co-reactant that is an amine or amine component (preferably TPA) that forms a strong reducing agent when oxidized;
(Iii) released by an assay composition comprising a component selected from the group consisting of the ECL component and the amine or an electrolyte in which the amine component can function as a medium in which it can be oxidized.

Another embodiment of the present invention is:
(A) a phosphate-specific antibody;
(B) a reagent selected from the group consisting of phosphorylase, a substrate of phosphorylase, or a combination thereof;
And (b) a composition containing a pH buffer and substantially free of inorganic phosphate, preferably free of inorganic phosphate. The composition preferably also includes an ECL coreactant (eg, TPA).

5.8 Kits One aspect of the invention relates to a kit comprising one or more components of an ECL assay buffer of the invention in one or more containers. Those components can optionally be combined with additional reagents to form an ECL assay buffer of the invention. The kit can also include additional assay related components such as ECL labels, ECL labeled assay reagents, enzymes, binding reagents, electrodes, assay plates and the like.

  Another aspect of the present invention relates to a kit comprising one or more ECL assay buffers comprising a trialkylamine co-reactant of the present invention other than TPA in one or more containers. The kit is preferably contained in one or more glass or plastic containers appropriately labeled with buffer content and information regarding proper storage and use in the assay. Some or all of the ECL assay buffer components can be stored dry. The kit may further include other assay related components such as enzymes, binding reagents, electrodes, assay plates.

  Another aspect of the present invention is to contain in one or more containers substantially free of inorganic phosphate and / or an ECL assay buffer reducing agent (preferably a non-phosphate pH buffer). To a kit comprising one or more ECL assay buffers. The kit is preferably contained in one or more glass or plastic containers appropriately labeled with buffer content and information regarding proper storage and use in the assay. Some or all of the ECL assay buffer components can be stored dry. The kit can further include other assay related components such as ECL labels, ECL labeled assay reagents, enzymes, binding reagents, electrodes, assay plates.

  Formal studies on the storage stability of Gly-Gly / TPA buffer have not been conducted. However, using assay buffer after 3-4 months had no effect on assay performance. Applicants believe that the same precautions should be taken for Gly-Gly stability as for example ORIGEN assay buffer. The concentration of divalent ions in the solution is preferably kept below the μM level.

  The kit is preferably configured or suitable for performing an ECL assay that detects electromagnetic radiation emitted from the composition. The kit includes a pH buffer in one or more containers, and preferably is substantially free of inorganic phosphate and / or an ECL assay buffer reducing agent (preferably a non-phosphate pH. Buffer). The kit includes (i) a metal-containing ECL component that is convertible to an excited state from which electromagnetic radiation is emitted, and (ii) an ECL co-reactant (preferably an amine or amine) that forms a strong reducing agent when oxidized. Component) and (iii) an electrolyte capable of functioning as a medium in which the ECL component and the ECL co-reactant (eg, amine or amine component) can be oxidized therein. Other ingredients are also included. The kit includes at least one single component comprising one or more components of the group consisting of the ECL component (i), ECL coreactant (ii), and electrolyte (iii).

  Another aspect of the invention relates to kits used to perform assays, preferably luminescence assays, more preferably electrode induced luminescence assays, most preferably electrochemiluminescence assays. The kit includes, in one or more containers, one or more pH buffering agents substantially free of inorganic phosphate, and (a) at least one luminescent label (preferably an electrochemiluminescent label), (B) at least one ECL co-reactant, (c) one or more phosphate-specific binding reagents, (d) one or more electrodes and / or magnetic beads, (e) one or more blockings. At least one assay selected from the group consisting of drugs, (f) preservatives, (g) stabilizers, (h) enzymes, (i) detergents, (j) desiccants, and / or (k) hygroscopic agents. And ingredients.

  The kits are each filed on June 28, 2002, and are incorporated herein by reference, “Assay Plates, Reader Systems and Methods for Luminescence Test Measurements”. One or more assay electrodes, preferably assay plates, more preferably multi-well assay plates, and one or more, according to U.S. patent application Ser. Nos. 10 / 185,274 and 10 / 185,363. Preferably, an assay module is included that comprises assay components in preferably two or more, more preferably three or more containers.

  According to one embodiment, the kit includes one or more assay components in one or more multi-well plate wells, preferably in a dried form.

  According to one embodiment, each assay component is in a separate container. According to another embodiment, the kit includes a container containing a binding reagent and a stabilizer. According to another embodiment, the well reagent may include a binding reagent, a stabilizer. The kit preferably contains no liquid in the well.

  One preferred embodiment comprises an assay plate, preferably a multiwell assay plate, one or more pH buffering agents, at least one luminescent label (preferably an electrochemiluminescent label) and at least one electrochemiluminescent co-reaction. The invention relates to a kit for use in performing an electrode induced luminescence assay (preferably an electrochemiluminescence assay) comprising at least one assay component selected from the group consisting of: The pH buffer includes an ECL assay buffer background reducing agent (preferably a non-phosphate pH buffer) or is substantially free of phosphate and / or the ECL co-reactant is TPA. (Preferably a functional tertiary alkylamine, most preferably PIPES).

  Another embodiment includes a multi-well plate, a pH buffer, at least one electrode-induced luminescent label (preferably an electrochemiluminescent label), and / or at least one bioreagent, and / or at least one blocking agent. (For example, BSA). The kit includes an ECL assay buffer background reducing agent (preferably a non-phosphate buffer), is substantially free of inorganic phosphate, and / or an ECL co-reactant other than TPA ( Preferably functional tertiary alkyl amines, most preferably PIPES) are included.

  According to a preferred embodiment, the kit comprises a complete cell, cell lysate, cell fragment, cell membrane, membrane ghost, organelle, organelle fragment, organelle membrane, virion, virion fragment, virion membrane , Liposomes, detergent-soluble proteins, detergent-soluble lipids, or combinations thereof.

  According to another embodiment, the kit includes plasma membrane fragments, endosomes, clathrin-coated vesicles, endoplasmic fragments, synaptic vesicles, Golgi fragments, membrane subdomains, mitochondria, peroxisomes, It consists of lysosomes, liposomes, virus particles, particles isolated from cells and encapsulated in membranes induced by viruses (lipid-derived membrane cells) and complete membrane lipid bodies A biomaterial selected from the group is included.

  According to one preferred embodiment, the kit comprises an antibody, antibody fragment, protein, enzyme, enzyme substrate, inhibitor, cofactor, antigen, hapten, lipoprotein, liposaccharide, cell, subcellular component, cell receptor , Preferably immobilized on a plate surface selected from viruses, nucleic acids, antigens, lipids, glycoproteins, carbohydrates, peptides, amino acids, hormones, protein binding ligands, drugs, luminescent labels (preferably ECL labels) or combinations thereof. At least one bioreagent. Preferably, the at least one bioreagent is made or selected to bind to one or more membranes, resulting in an electrode having such an immobilized membrane.

Biomaterials include single transmembrane receptors with tyrosine kinase intrinsic activity; non-tyrosine kinase transmembrane receptors (eg, transferrin receptor); G-protein coupled receptors (GPCR); GPCR effector proteins (eg, adenyl) Acid cyclase); phosphoinositides (eg, phosphatidylinositol 4,5 bisphosphate (PIP ₂ )); phospholipid or sphingolipid composition, identification or function (ie, phosphatidyl during apoptosis) Changes in the presence of serine); GTP hydrolase / guanine nucleotide exchange factor (GEF) / GTP hydrolase activating protein (GAP) bound to organelles; Cellular adhesion molecules (eg, VCAM, integrin); cytoplasmic peripheral membrane protein kinases (eg, src); intracellular protein kinase adapter / docking protein (eg, insulin receptor substrate) 1, GRB2); ion channels (eg, nicotine acetylcholine receptor, CFTR, etc.); passive transporters (eg, glucose); active (ATP-driven) transporters; ion-linked transporters (eg, Na + Glycosyltransferase / glycoprotein modifying enzyme; nuclear membrane fragment; and at least one active protein selected from the group consisting of soluble receptors It is preferred to include a lipid / protein layer.

  The kit preferably includes an immobilization reagent comprising a protein, nucleic acid or combination thereof.

  According to one preferred embodiment, the plurality of wells contain at least two different bioreagents. For example, one well can contain two or more assay domains containing different bioreagents.

  Preferably, the kit comprises at least one electrochemiluminescent co-reactant and / or at least one electrode induced luminescent label (preferably an electrochemiluminescent label).

  According to another embodiment, the kit is compatible with multiple assays. Preferably, the kit further comprises additional assay reagents used for additional assays selected from the group consisting of radioactivity assays, enzyme assays, chemical colorimetric assays, fluorescent assays, chemiluminescent assays and combinations thereof. .

  According to another embodiment, the kit comprises 2 or more, preferably 4 or more, more preferably 8 or more, more preferably 15 or more, most preferably 25 or more assay modules or plates. It is. According to a preferred embodiment, the kit is contained in a bag or container (eg, ziplock) that can be sealed any number of times.

  The bag or container is preferably substantially impermeable to water. According to one embodiment, the bag is preferably a metal foil, preferably an aluminized foil.

  The packaging can be translucent, transparent or opaque. The plate is preferably packaged in a plastic container or bag containing a dry or inert atmosphere lined with aluminum (eg, the bag can be sealed under a nitrogen or argon atmosphere, or a desiccant is applied to the bag). Can be included). According to another embodiment, the container is vacuum sealed.

  The container preferably contains one plate. According to another embodiment, the container includes 10 plates. According to another embodiment, the container contains 10-100 plates.

  The assay module or plate is preferably sterile and / or substantially free of dust and other contaminants.

  The assay module is also preferably substantially sterile.

  According to one embodiment, the kit is manufactured and / or packaged (at least partially) in a “clean room” environment. The kit is manufactured (and / or at least partially) in a class 100,000 clean room of <100,000 particles (clean room particle count with 0.5 micron particle count) / cubic foot (35.3 million particles / cubic meter). Preferably it is packaged.

  Preferably, the kit has a contaminating particle count (particles less than 0.5 microns) of less than 60 million per cubic meter, more preferably 30 million per cubic meter, even more preferably less than 20 million, even more preferably less than 15 million, most Preferably it is less than 10 million.

Preferably, non-volatile residue in deionized water is less than 0.50 g / ^{m 2,} more preferably less than 0.25 g / ^{m 2,} even more preferably less than 0.15 g / ^{m 2,} most preferably 0.10 g / less than m ² .

  Preferably, the concentration of contaminating ions is less than 50 ppm, more preferably less than 20 ppm, even more preferably less than 10 ppm, even more preferably less than 5 ppm, and most preferably less than 1 ppm.

5.9 Methods Another aspect of the present invention relates to methods of using the improved buffers, reagents and / or compositions of the present invention.

  One embodiment of the invention relates to a method of performing an electrochemiluminescence assay in which electrochemiluminescence is induced in the presence of an ECL assay buffer of the invention. Electrochemiluminescence is preferably induced using a carbon-based electrode.

  Another embodiment of the invention relates to a method for measuring the amount of ECL label. The ECL label is induced in the presence of the ECL assay buffer of the present invention to emit electrochemiluminescence, and the amount of ECL label is measured by measuring the electrochemiluminescence. Electrochemiluminescence is preferably induced using a carbon-based electrode. Most preferably, the ECL label is bound and retained near the electrode.

  Another embodiment of the invention relates to a method for measuring the amount or activity of an analyte. The analyte reacts with the labeling substance containing the ECL label, forms a complex with the labeling substance containing the ECL label, or competes in a specific binding interaction with the labeling substance containing the ECL label. The ECL label is induced in the presence of the ECL assay buffer of the present invention to emit electrochemiluminescence and the electrochemiluminescence is measured to determine the amount or activity of the analyte. Electrochemiluminescence is preferably induced using a carbon-based electrode. Most preferably, due to the presence or activity of the analyte, the label is bound to or released from the electrode (eg, through formation of a specific binding complex or through cleavage or formation of a chemical bond). .

  One embodiment of the invention relates to a method of performing an electrochemiluminescence assay. Electrochemiluminescence is induced in the presence of a composition comprising a pH buffer and an ECL co-reactant, said composition being substantially free of inorganic phosphate and / or an ECL assay buffer background reducing agent. (Preferably a non-phosphate pH buffer).

  Another embodiment of the invention relates to a method of performing an electrochemiluminescence assay. Electrochemiluminescence is induced in the presence of a composition comprising a pH buffer and an ECL coreactant. The ECL coreactant is a functional trialkylamine, preferably PIPES.

Another embodiment of the present invention is a method for causing electromagnetic radiation emission comprising:
(A) (i) a metal-containing ECL component that is convertible to an excited state from which electromagnetic radiation is emitted;
(Ii) an ECL co-reactant (preferably an amine or amine component) that forms a strong reducing agent when oxidized;
(Iii) an electrolyte capable of functioning as a medium in which the ECL component and the ECL co-reactant (eg, an amine or amine component) can be oxidized;
(Iv) forming a composition comprising a pH buffer, wherein the composition is substantially free of inorganic phosphate and is an ECL background reducing agent (preferably a non-phosphate pH buffer And / or the ECL co-reactant is a functional tertiary alkyl amine;
(B) exposing the composition under conditions suitable for an amount of electrochemical energy effective to induce the composition to emit electromagnetic radiation;
(C) detecting the emitted electromagnetic radiation;

  Another embodiment of the invention is a method for qualitatively or quantitatively performing a specific binding assay in a sample or composition comprising a pH buffer substantially free of inorganic phosphate and phosphate-specific antibodies. About. It is preferred that the sample or composition further comprises an ECL co-reactant.

  Another embodiment of the present invention employs a composition comprising a pH buffer substantially free of inorganic phosphate in a well comprising one or more assay domains onto which a binding reagent is immobilized. , To a method of performing a specific binding assay qualitatively or quantitatively. Preferably, the composition further comprises a phosphate specific antibody.

  Another embodiment of the present invention is a functional trialkylamine ECL that is substantially free of inorganic phosphate and / or in a well comprising one or more assay domains on which binding reagents are immobilized. It relates to a method for qualitatively or quantitatively performing a specific binding non-washing assay using a composition comprising an ECL assay buffer comprising a co-reactant.

  Another embodiment of the invention relates to a method of performing an assay comprising the step of forming a complex comprising a kinase product and a phosphate-specific antibody, wherein the complex is not exposed to inorganic phosphate.

Another embodiment of the present invention is:
(A) forming a complex comprising a kinase product and a phosphate-specific antibody, wherein the complex is not exposed to inorganic phosphate;
(B) inducing a metal-containing ECL component to emit electromagnetic radiation;
And (c) detecting the emitted electromagnetic radiation.

  It is preferable that the composite further contains the metal-containing ECL component.

Another embodiment of the present invention is:
(A) a metal-containing ECL component that contains a pH buffer and is substantially free of inorganic phosphate, (i) is convertible to an excited state in which electromagnetic radiation is emitted, (ii) strong reduction when oxidized. Forming a composition comprising an amine or amine component that forms an agent, and / or (iii) the ECL component and an electrolyte that can function as a medium in which the amine or amine component can be oxidized;
(B) exposing the composition to an amount of electrochemical energy effective to induce the composition to emit electromagnetic radiation under appropriate conditions;
And (c) detecting the emitted electromagnetic radiation.

  Another aspect of the invention relates to an improved assay. The present invention is useful, for example, to allow detection and / or quantification of one or more target analytes. These reactions include, for example, antigen-antibody interactions, ligand-receptor interactions, DNA and RNA interactions, enzymatic reactions, and other known reactions. In certain embodiments, the present invention relates to a method for qualitatively and quantitatively detecting the presence of a target analyte in a multicomponent sample or multicomponent system. (U.S. Patent Application No. Glezer et al., Filed on the same day as the present application and incorporated herein by reference (Title: “Methods and Apparatus for Conducting Multiple Measurements on a Sample (Methods and Apparatus for Conducting Multiplexing)”). See also Measurements on a Sample) [Attorney Reference Number 100405-06410]).

  A preferred embodiment of the present invention includes (a) a phosphate-specific antibody, (b) an assay comprising a capture reagent that is immobilized on a solid surface comprising or adjacent to an electrode, and / or (c) A method comprising one or more of the assays comprising a low detection level (and / or requiring a high signal / background ratio).

  Embodiments of the present invention can be used to test a variety of samples that may contain the analyte of interest or activity. Such a sample can be in the form of a solid, emulsion, suspension, liquid or gas. These include, for example, (live or dead) cells and products derived from cells, immortalized cells, cell fragments, cell fractions, cell lysates, cell organelles, cell membranes, hybridomas (antibody producing organisms such as hybridomas). Cell culture supernatant, waste water or drinking water, food, beverage, pharmaceutical composition, blood, serum, plasma, hair, sweat, urine, feces, tissue, biopsy sample, effluent , Separation and / or fractionation sample, separation and / or fractionation liquid, organ, saliva, animal part, animal by-product, plant, plant part, plant by-product, soil, mineral, deposit, water, water supply, water source , Fluid (gas and liquid) filtration residue, beers (swipes), absorbent material, gel, cytoskeleton, protein complex, unfractionated sample, unfractionated cell lysate, endocrine factor, paracrine factor, autocrine factor , Cytokines, hormo , Cell signaling factor and / or component, second messenger signaling factor and / or component, cell nucleus, nuclear fraction, chemical substance, chemical solution, structural biological component, skeletal (ligament, tendon) component, separation and / or fraction Painting skeleton component, hair, fur, feathers, hair fraction and / or isolate, skin, skin sample, skin fraction, dermis, endothelium, eukaryotic cell, prokaryotic cell, fungus, yeast, antibody, antibody fragment, immune factor , Immune cells, drugs, therapeutic drugs, oils, extracts, mucus, furs, oils, sewage, environmental samples, samples containing organic solvents or air, or samples derived therefrom. It is not limited. The sample can further include, for example, water, an organic solvent (eg, acetonitrile, dimethyl sulfoxide, dimethylformamide, n-methylpyrrolidone, or alcohol) or a mixture thereof.

  Analytes that can be measured include whole cells present in the sample, cell surface antigens, subcellular particles (eg, organelles or membrane fragments), viruses, prions, dust mites or fragments thereof, viroids, antibodies, antigens , Hapten, fatty acid, nucleic acid (and synthetic analog), protein (and synthetic analog), lipoprotein, polysaccharide, inhibitor, cofactor, hapten, cell receptor, receptor ligand, lipopolysaccharide, glycoprotein, peptide, polypeptide , Enzyme, enzyme substrate, enzyme product, second messenger, cellular metabolite, hormone, drug, synthetic organic molecule, organometallic molecule, tranquilizer, barbiturate, alkaloid, steroid, vitamin, amino acid, sugar, lectin, recombinant Or derived protein, biotin, avidin, streptavidin, inorganic content And the like, but is not limited only to these. The activities that can be measured include phosphorylase, phosphatase, esterase, transglutaminase, nucleic acid damaging activities, transferase, oxidase, reductase, dehydrogenase, glycosidase, ribosome, protein processing enzyme (eg, protease, kinase, Protein phosphatase, ubiquitin-protein ligase, etc.), nucleic acid processing enzymes (eg, polymerase, nuclease, integrase, ligase, helicase, telomerase, etc.), cell receptor activation, second messenger system activation, etc. It is not limited to only.

  The whole cell may be an animal, plant or bacterium and may be alive or dead. Examples include plant pathogens such as fungi and nematodes. The term “subcellular particle” refers to, for example, subcellular organelles, membrane particles of divided cells, cell wall fragments, ribosomes, complexes of multiple enzymes, and other particles that can be derived from living organisms. Is included. Nucleic acids include, for example, chromosomal DNA, plasmid NA, viral DNA, and recombinant DNA derived from multiple sources. Nucleic acids also include RNA, such as messenger RNA, ribosomal RNA, and transfer RNA. Polypeptides include, for example, structural proteins such as enzymes, transport proteins, receptor proteins, and viral coat proteins. Preferred polypeptides are enzymes and antibodies. A particularly preferred polypeptide is a monoclonal antibody. Examples of hormones include insulin and T4 thyroid hormone. Examples of drugs include cardiac glycosides. It is of course within the scope of the present invention to include synthetic polypeptides, synthetic nucleic acids, and synthetic substances that are chemically similar to biological materials such as synthetic membranes, vesicles and liposomes. The above is not a comprehensive list of biological materials suitable for use in the present invention, but merely to illustrate the broad scope of the invention.

  The composition or reagent of the present invention is preferably aqueous. The composition or reagent can also be non-aqueous. Examples of suitable organic liquids are acetonitrile, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), methanol, ethanol, and mixtures of two or more of the above. As an example, tetraalkylammonium salts such as tetrabutylammonium tetrafluoroborate are soluble in organic liquids and can be used with organic liquids to form non-aqueous electrolytes.

Also, generally, the target analyte is present at a concentration of 10 ⁻³ mol or less, for example at least 10 ⁻¹⁸ mol. Samples that can contain the analyte of interest can be in the form of solids, emulsions, suspensions, liquids or gases, eg cells and products induced by cells, water, food, blood, serum, hair It can be derived from sweat, urine, feces, tissue, saliva, oil, organic solvents or air. The sample can further comprise, for example, water, acetonitrile, dimethyl sulfoxide, dimethylformamide, n-methyl-pyrrolidone or alcohol.

  In one embodiment, the reagent is an antibody, antigen, nucleic acid, hapten, small nucleotide sequence, oligomer, ligand, enzyme, or biotin, avidin, streptavidin, protein A, protein G, or a complex thereof, or Such as an ECL component complexed to another second binding partner that can bind to the first binding partner by protein interaction.

One embodiment of the invention relates to a method for detecting or quantifying a target analyte by an ECL assay. This method
(1) (a) a sample to be tested for the target analyte;
(B) (i) an additional target analyte or analog of the target analyte;
(Ii) the target analyte or its analog binding partner;
(Iii) at least one substance selected from the group consisting of (i) or (ii) and a reactive component capable of binding;
(C) can be converted into an excited state from which electromagnetic radiation is emitted and binds to (b) the target analyte or the substance defined in (b) (i), (b) (ii) or (b) (iii) A metal-containing ECL component capable of interacting;
(D) an ECL co-reactant (preferably an amine or amine component) that forms a strong reducing agent when oxidized;
(E) forming a composition comprising the ECL component and an electrolyte capable of functioning as a medium in which the species can be oxidized;
(2) exposing the composition to an amount of electrochemical energy effective to induce the composition to emit electromagnetic radiation;
(3) detecting the emitted electromagnetic radiation, wherein the sample is not exposed to inorganic phosphate that is detrimental to assay performance, and the composition further comprises an ECL assay buffer background reducing agent (preferably non- Phosphate pH buffer). Preferably, the composition comprises less than 15 mM inorganic phosphate, more preferably less than 5 mM inorganic phosphate, even more preferably less than 1 mM phosphate, and even more preferably substantially comprises inorganic phosphate. And most preferably free of inorganic phosphate.

  In solid phase assay formats (eg, solid phase binding assays), biological activity or binding reactions are often combined with the attachment of labels to the surface or the dissociation of labels from the surface. For example, the binding interaction between the attached binding reagent and the labeled analyte results in localization of the label on the solid phase supporting the immobilized binding reagent. The biological activity or binding reaction to be measured can be quantified by measuring the label on the solid phase. Many solid phase assay formats include a wash step that removes unbound label before detecting the label on the solid phase (wash assay). An assay that does not include this washing step can be performed when the detection method can distinguish between free and bound labels. A non-wash assay format is desirable because the wash step can be difficult or laborious to perform in many applications. However, in many cases, unwashed assay performance is limited due to the high background signal due to incomplete discrimination between free and bound labels.

  Applicants have surprisingly found that in the ECL assay buffer of the present invention, in the ECL assay, the working electrode used to induce ECL (eg, covalent interaction, specific binding interaction, non-specificity). It has been found that assay reagents attached (such as by mechanical adsorption) improve the discrimination between free and bound labels (ie, the ability to selectively detect the label bound to the electrode). More specifically, the compositions and reagents of the present invention improve the ratio of ECL signal from bound label to ECL signal from free label. It is believed that the ECL assay buffer of the present invention reduces the distance from the solid electrode surface where ECL labeling is induced to produce luminescence. This increases the signal of the bound label (bound to the electrode surface) relative to the free label (not bound to the electrode). Another way of characterizing this is by "effective excitation length" (the maximum distance at which free ECL label can be excited). “Effective excitation length” refers to i) the distance that these short-lived intermediates involved in ECL generation (eg, oxidation products of TPA) can diffuse from the electrode before they are destroyed by destructive side reactions (these And ii) the rate at which the free label or labeling reagent diffuses to a region sufficiently close to the electrode to participate in the reaction with these reactive intermediates (unbound ECL label). Or a function of the diffusion constant of the labeling reagent).

  With the ECL assay buffer of the present invention, the effective excitation length is reduced by> 50%, preferably> 75%, even more preferably> 90%. Accordingly, ECL assay buffers of the invention are desirable because they maximize the bound / free ECL signal ratio that improves assay performance. These considerations require the presence of a high concentration of labeled species in the solution, but measure low affinity interactions where significant signal loss is expected due to the dissociation of the binding complex during the wash step. It is especially important when doing so.

  Accordingly, another aspect of the invention relates to a non-washing format assay that is substantially free of pH buffering inorganic phosphate and maximizes the binding / free ECL signal ratio. The assay preferably captures ECL label on the surface including or adjacent to the electrode surface. For example, U.S. Patent Nos. 6,066,448, 6,090,545, 6,140,045, 6,207,369, 6,214,369, and US patent application entitled “Assay Plates, Reader Systems and Methods for Luminescence Test Measurements,” filed on June 28, 2002 and incorporated herein by reference. See 10 / 185,274 and 10 / 185,363.

  Accordingly, another embodiment of the present invention generally relates to an electrochemiluminescent assay that uses reagents immobilized on a surface (preferably an electrode surface) and has an advantageous effective excitation length. Preferably, in this assay, the effective excitation length is less than 100 microns, more preferably less than 75 microns, even more preferably less than 50 microns, even more preferably less than 25 microns, even more preferably less than 10 microns, even more preferably. Less than 5 microns, most preferably less than 1 micron. According to a particularly preferred embodiment, the effective excitation length is less than 0.5 microns, preferably less than 0.2 microns, and even more preferably less than 0.1 microns.

5.10 System Yet another aspect of the present invention relates to a system comprising or using a reagent and / or composition of the present invention for performing an assay.

One embodiment of the invention relates to a system for ECL detection or quantification of a target analyte in a sample. The system
(A) a pH buffer; (b) a sample;
(C) (i) the added target analyte or analog of the target analyte;
(Ii) the target analyte or its analog binding partner;
(Iii) at least one substance selected from the group consisting of (i) or (ii) and a reactive component capable of binding, wherein one of said substances is convertible to an excited state from which electromagnetic radiation is emitted A substance linked directly or via one or more other molecules to a metal-containing ECL component which is
(D) a strong reducing agent and an ECL co-reactant that is convertible to an electrolyte, preferably an amine or an amine component;
(D) one or more electrodes that induce ECL components to emit electromagnetic radiation;
(E) one or more detectors for measuring the emitted radiant energy to determine the presence or amount of the analyte of interest in the sample;
The pH buffer is a substantially phosphate free ECL assay buffer background reducing agent and / or the ECL co-reactant is a functional tertiary amine.

Another embodiment of the invention relates to a system for ECL detection or quantification of an analyte of interest in a sample. The system
(A) a pH buffer; (b) a sample;
(C) (i) the added target analyte or analog of the target analyte;
(Ii) the target analyte or its analog binding partner;
(Iii) at least one substance selected from the group consisting of (i) or (ii) and a reactive component capable of binding, wherein one of said substances is convertible to an excited state from which electromagnetic radiation is emitted A substance linked directly or via one or more other molecules to a metal-containing ECL component which is
(D) a strong reducing agent and an ECL co-reactant that is convertible to an electrolyte, preferably a functional tertiary amine;
(D) one or more electrodes that induce ECL components to emit electromagnetic radiation;
(E) one or more detectors for measuring the emitted radiant energy to determine the presence or amount of the analyte of interest in the sample.

11 Method of Selecting a Bioactive Compound and Producing a Novel Drug Another aspect of the present invention is the “assay plate, reader for luminescent test measurement, filed on June 28, 2002, each of which is incorporated herein by reference. As described in paragraph 6.11 of U.S. Patent Application Nos. 10 / 185,274 and 10 / 185,363 entitled "Systems and Methods (Assay Plates, Reader Systems and Methods for Luminescence Test Measurements)", It relates to improved methods and systems for selecting or identifying bioactive compounds and, in some cases, incorporating such bioactive compounds into a suitable carrier composition at a suitable dose.

  One embodiment preferably includes more than 50, more preferably 100, more preferably 500, even more preferably 1,000, most preferably 5,000 screening, preferably high throughput screening ( HTS) relates to the use of the invention for screening. According to particularly preferred embodiments, the screening comprises more than 10,000, more than 50,000, more than 100,000, more than 500,000 and / or more than 1,000,000 compounds.

  The reagents, compositions, methods, devices and / or assay plates or modules of the present invention are advantageously incorporated into and / or used in various screening and / or drug discovery methods. Such screening and / or drug discovery methods include Blake US Pat. No. 5,565,325, Chen et al US Pat. No. 5,593,135, Thrustrup et al US Pat. No. 5,521,135. Agrafiotis et al., US Pat. No. 5,684,711, Dower et al., US Pat. No. 5,639,603, Ashby et al., US Pat. No. 5,569,588, US Pat. No. 5,541,061, US Pat. No. 5,574,656 and the method shown in US Pat. No. 5,783,431 to Peterson et al.

  According to another embodiment, the present invention further includes the steps of identifying side effects associated with the drug and storing information about the side effects in a database. See Classen US Pat. No. 6,219,674.

  Another aspect of the present invention relates to improved bioactive compounds and / or drugs prepared using the methods of the present invention.

(Example)
The following examples illustrate some of the electrodes, plates, kits and methods that are within the scope of the present invention. It goes without saying that these examples are not to be construed as limiting the invention in any way. Numerous variations and modifications of the present invention can be made without undue experimentation by those skilled in the art.

ECL Measurements ECL measurements were performed using multiwell plates with integrated carbon ink electrodes unless otherwise indicated (Emission Test Measurements, each filed on June 28, 2002 and incorporated herein by reference). Example of co-pending US patent applications 10 / 185,274 and 10 / 185,363 entitled "Assay Plates, Reader Systems and Methods for Luminescence Test Measurements" 6.1 and in particular plate types A and B). The electrodes were optionally treated with oxygen plasma before being coated with the binding reagent (hereinafter, plasma treated and non-plasma treated plates are referred to as PT or NPT plates, respectively). The binding reagent was immobilized on the working electrode of the plate using the methods described in US patent applications 10 / 185,274 and 10 / 185,363, or modifications thereof. Unless indicated otherwise, ECL measurements were performed using a multiwell plate reader configured to use these multiwell plates. Readers and their uses are described in Example 6.3 of US patent application Ser. Nos. 10 / 185,274 and 10 / 185,363. US patent application Ser. Nos. 10 / 185,274 and 10 / 185,363 and, in particular, descriptions of plate types, immobilization methods, plate readers and ECL measurement methods are incorporated herein by reference. The reported ECL intensity is a relative value and may depend on the instrument, gain setting and plate used in the individual experiment. For this reason, the absolute values reported in different experiments cannot be directly compared.

The tyrosine kinase assay consists of phosphorylation of a kinase substrate immobilized on an electrode in a multiwell plate configured for ECL measurement (see Example I), complexation of the product to a labeled anti-phosphotyrosine antibody, and Detection of surface bound label by ECL measurement in the presence of ECL assay buffer containing ECL co-reactant is included. The electrode was pre-etched with oxygen plasma to increase the amount of exposed carbon. A kinase substrate of poly (Glu, Tyr) (PGT, Sigma-Aldrich Co.) having a Glu to Tyr ratio of 4: 1 and a molecular weight of 20,000 to 50,000 daltons is added to the plate wells. Non-specifically adsorbed on the electrode surface and fixed. The working electrode in each well was treated with 1500 nL of a solution containing 1 mg / ml PGT diluted with PBS buffer. The plates were then dried overnight and blocked at 4 ° C. in a 5% solution of bovine serum albumin. The plate was washed to remove the blocking agent before use.

Anti-phosphotyrosine monoclonal antibody (Abzyme, IGEN International) labeled with soluble tyrosine kinase (c-src, Upstate Biotechnology), sulfonated derivative of ruthenium-tris-bipyridine (Sulfo-TAG ™ label, Meso Scale Discovery), ATP And the assay was performed by adding 50 μL of a buffer solution containing Mg ⁺² to each well. The reaction was allowed to proceed for 1 hour. The plate was washed and 100 μL of ECL assay buffer containing tripropylamine was added. Plates were analyzed by electrochemiluminescence detection as described in Example 1.

  When a conventional ECL assay buffer (ORIGEN Assay Buffer, IGEN International) containing TPA diluted in phosphate buffer is used in this procedure, the complex formed between the labeled antibody and the phosphorylated substrate is Dissociation took 30 to 1 hour due to competitive binding of ions and labeled antibody (most of the dissociation occurred in the first few minutes). One approach to avoiding this problem is to control the time that the formed complex is exposed to the inorganic phosphate-containing solution. However, this approach may not be feasible for some assays, such as high throughput assays involving a large number of plates.

  Applicants have discovered that another solution to overcome this problem is to use a phosphate-free buffer. Surprisingly, it has been discovered that phosphate can be replaced with other buffers without compromising the ability of the ECL assay buffer to help generate ECL.

The assay was performed using the following two ECL assay buffer compositions.
Gly-Gly assay buffer:
0.4M glycylglycine (Gly-Gly)
0.1M tripropylamine (TPA)
12 mM ethylenediaminetetraacetic acid (EDTA)
pH = 7.8 (adjusted by adding 10% KOH)
Tris assay buffer:
0.4M Tris (hydroxymethyl) aminomethane (Tris)
0.15M tripropylamine (TPA)
12 mM ethylenediaminetetraacetic acid (EDTA)
pH = 7.8 (adjusted by adding 10% KOH)

The phosphorylation reaction was stopped by adding EDTA to a new ECL assay buffer to sequester Mg ⁺² , an ion required for kinase activity (the phosphate has a high affinity for magnesium ions, so EDTA was not required for the salt-based ECL assay buffer). This composition allowed two steps (addition of stop solution and actual assay buffer) to be combined into one step. Applicants have found that EDTA interferes with ECL generation at concentrations higher than 10 mM, but 5-12 mM EDTA is sufficient to stop the reaction with only a slight reduction in ECL signal.

  FIG. 1 shows the decrease in ECL from the phosphopeptide-antibody complex as a function of time from addition of assay buffer to ECL measurement. Surprisingly, the complex dissociated almost completely when exposed to phosphate-containing ORIGEN assay buffer (within the 0 minute time required to place the plate in the ECL reader), but Gly-Gly. Excellent stability in assay buffer (<80% dissociation at 40 minutes) and Tris (<55% dissociation at 40 minutes) assay buffer.

  The stability of the complex was further improved by omitting the washing step prior to adding the assay buffer. In FIG. 2, 200 μL of Gly-Gly assay buffer was added directly to the reaction mixture without the wash step (as described above except that the EDTA concentration was 5 mM and 0.2% Triton X-100 was added). The result of an experiment is shown. Saving the plate for 20 hours before measuring ECL only reduced the signal by 15%.

  One of the more surprising advantages of this procedure was its robustness. Surprisingly, the time of the phosphorylation step was the only time that had to be tightly controlled to obtain reproducible results. The assay results were independent of the time between all other steps.

Detection of phosphorylated EGF receptor Autophosphorylated α-epidermal growth factor receptor (α-) in cell lysates prepared from EGF activated A-431 cells (American Type Culture Collection) using a sandwich immunoassay. EGFR) was detected. This assay used a biotin-labeled capture antibody against the α-EGFR extracellular domain and a Sulfo-TAG labeled detection antibody specific for phosphotyrosine (see Example 2). The biotin-labeled antibody was immobilized on the working electrode of a multiwell plate configured for use in an ECL assay (see Example 1) by the interaction between the biotin label and avidin passively adsorbed on the electrode surface. Then solubilized EGFR (in RIPA, deoxycholate containing buffer) was added and allowed to bind to the anti-EGFR antibody. Subsequently, Sulfo-TAG labeled α-phosphotyrosine antibody was added to detect autophosphorylated EGFR.

  Prior to conducting the assay as described above and inducing and measuring ECL in the final product stability experiments, the resulting sandwich complex was transformed into two different ECL assay buffers, namely 150 mM TPA / 150 mM phosphorous. Incubated at different times in acid salts, pH 7.48 and 100 mM TPA / 400 mM glycine-glycine, pH 7.8. FIG. 3 shows that the signal is significantly attenuated in the presence of phosphate containing buffer as a function of time. That is, the signal decreased by approximately 80% after 1 hour. With glycine-glycine buffer this reduction was suppressed to approximately 20%. The signal is greatly decreased by the phosphate buffer because the phosphate ion competes with the phosphorylated protein for the anti-phosphotyrosine antibody. The signal / background ratio was also increased 2.5-fold by glycyl-glycine assay buffer.

Effect of ECL Assay Buffer Composition on Specific Signal and Assay Buffer Background Discrimination Ability In many ECL assay formats, the sensitivity of measuring ECL label is generated by ECL assay buffer in the absence of ECL label Limited by light signal (and noise in light signal) (ECL assay buffer background). This limitation is particularly evident in wash assays that use ECL readers with low non-specific binding levels and / or with high sensitivity, low noise photodetectors. Applicants examined the relationship between the ECL assay buffer composition and the ability to discriminate between the signal from the ECL label and the ECL assay buffer background. In particular, Applicants have identified four ECL assay buffer formulations with varying ECL co-reactant type and / or pH buffer type: TPA / phosphate, TPA / Tris, TPA / Gly-Gly and PIPES / phosphate (where PIPES represents 1,4-piperazine-1,4-bis (representing 2-ethanesulfonic acid)).

  Experiments were performed on multiwell plates (described in Example 1) containing avidin immobilized on the working electrode. Experiments were performed on oxygen plasma treated plates (PT plates) and untreated plates (NPT plates). A 2.5 uL solution containing 0.5 mg / mL avidin and 0.0035% Triton X-100 is applied to the working electrode in each well, the solution is allowed to evaporate and allowed to dry for over 1 hour, and the remaining surface of the well is placed at 4 ° C. Avidin was immobilized on PT plates by blocking with 5% (w / v) BSA solution overnight. 2.5 uL of a solution containing 0.5 mg / mL avidin and 0.0075% Triton X-100 is applied to the working electrode of each well, the solution is evaporated to dry overnight, and the remaining surface of the well is 5% (w / V) Avidin was immobilized on NPT plates by blocking with BSA solution for 2 hours. By treating wells with a solution containing biotin-labeled bovine IgG and about 1.9 Sulfo-TAG labels per protein (BT-IgG *), various amounts of ECL near the electrode surface Incorporated signs. BT-IgG * was bound by adding 50 uL of PBS solution containing 1 nM BT-IgG * to the wells and incubating with shaking for 60 minutes. Wells were washed with water, 100 uL of ECL assay buffer was added, and ECL was measured. The above experiment was repeated except that BT-IgG * was omitted, and the signal due to ECL assay buffer background was measured.

  Optimize the four combinations of the co-reactant and pH buffer to be tested to give the best ratio of signal from BT-IgG * to ECL assay buffer background (S / B ratio), pH Buffer concentration and pH were identified. The concentration of the co-reactant was changed from 25 to 200 mM for TPA and from 13 to 200 mM for PIPES. The pH buffer concentration was varied from 50-300 mM for phosphate, 100-600 mM for Tris, and 50-800 mM for Gly-Gly. The pH was varied from 7-8. In all cases, ECL assay buffer also contained 0.05% Triton X-100. KOH or HCl was added as needed to reach the desired pH. Within the test range, all formulations showed sufficient performance for use in the ECL assay, but the following optimal formulation: TPA / phosphate (125 mM TPA, 200 mM phosphate, 0.05% Triton X-100, pH 7.5), TPA / Tris (125 mM TPA, 200 mM Tris, 0.05% Triton X-100, pH 7.8), TPA / Gly-Gly (100 mM TPA, 200 mM Gly-Gly, 0. 05% Triton X-100, pH 7.8) and PIPES / Phos (25 mM PIPES, 100 mM phosphate, 0.05% Triton X-100, pH 7.5) were identified based on their S / B ratio .

  Figures 4A and 4B compare the performance of the four optimal formulations for assays performed on NPT and PT plates, respectively. We have found that TPA / phosphate and TPA / Tris buffer give nearly equivalent signals to BT-IgG *, but the TPA / Tris system has a low ECL assay buffer background, so It has been found that the S / B ratio is significantly improved over the phosphate buffer. Assuming that background noise is approximately proportional to the background signal, a 4-6 fold improvement in S / B ratio is directly reflected in a 4-6 fold improvement in detection limit. Despite the low specific signal, TPA / Gly-Gly buffer has an S / B ratio that is about the same as that of TPA / Tris buffer, and therefore the detection limit is about the same. is expected. The PIPES / phosphate buffer was slightly better on the unetched plate and slightly worse on the etched plate than the TPA / phosphate buffer (with respect to the S / B ratio).

Effect of ECL assay buffer composition on the ability to discriminate between ECL label bound to the electrode surface and free in solution. Measurement of ECL label retained near the electrode in some ECL assay formats. Sensitivity is limited by the light signal (and light signal noise) generated by the ECL label in solution. This limitation is particularly evident in assays where addition of ECL assay buffer and washing prior to ECL measurement does not remove label in solution (non-washing assay). Applicants have determined the relationship between the ECL assay buffer composition and the ability to discriminate between signals due to the ECL label bound to (or held in proximity to) the electrode and the free ECL label in solution. Examined.

  In these experiments, the specific signal from the bound ECL label was measured using BT-IgG * bound to an avidin-coated electrode as described in Example 4. ECL assay buffer background was also measured without BT-IgG * as described in Example 4. From the free ECL label in solution, similar to the ECL assay buffer background, except that the ECL assay buffer added to the well contains 10 nM bovine IgG containing 3.9 labels per protein (IgG *). The ECL signal was measured. The ratio of the ECL signal from bound BT-IgG * to the ECL signal from free IgG * (B / F ratio) indicates the sensitivity with which the bound ECL label can be detected in the presence of free ECL label in solution. It is.

  The four ECL assay buffers optimized in Example 4 were tested for the ability to discriminate between surface bound ECL labels. The results are shown in Tables IA and IB. Replacing phosphate with Tris improved the B / F ratio in the case of TPA-containing buffers. However, the most extreme improvement was obtained by replacing the co-reactant component, ie, replacing TPA with PIPES. Replacing TPA / Phos with PIPES / Phos improved the B / F ratio by 4-5 times.

  PIPES-containing ECL assay buffer was prepared using phosphate, Tris or Gly-Gly as the buffer. The B / F ratio of each of these mixtures was further optimized by changing the PIPES concentration and buffer components. PIPES concentrations were varied from 12.5-200 mM for phosphate-based buffers and 25-100 mM for Tris and Gly-Gly buffers. The buffer concentration was varied from 100 to 400 mM. In all cases, ECL assay buffer also contained 0.05% Triton X-100. KOH or HCl was added as needed to reach the desired pH. Within the test range, all formulations, including compositions without added buffer component, performed well enough to be used for ECL assays. Since PIPES can act as a pH buffer, it is possible to obtain sufficient performance without adding a buffer. It was found that a PIPES concentration of 20-100 mM resulted in a high B / F ratio while maintaining reasonable ECL intensity. The best performance was obtained when the phosphate concentration was approximately 2-4 times the PIPES concentration. Based on the high S / B ratio and reasonable signal intensity, the following optimal formulation: PIPES / phosphate (25 mM PIPES, 100 mM phosphate, 0.05% Triton X-100, pH 7. 5), PIPES / Tris (25 mM PIPES, 200 mM Tris, 0.05% Triton X-100, pH 7.8), and PIPES / Gly-Gly (25 mM PIPES, 100 mM Gly-Gly, 0.05% Triton X-100) PH 7.8) was identified.

  Figures 5A and 5B compare the performance of the three optimal formulations for unwashed assays performed on NPT and PT plates, respectively. The figure compares performance against a conventional TPA / phosphate buffer. The inventors have shown that the B / F ratio is significantly improved (4-5 times) over TPA / phosphate by using PIPES as a co-reactant with the three buffers tested. I found it.

Effect of detergent on ECL assay buffer performance Figure 6 shows the effect of the presence or absence of various non-ionic detergents on the ECL signal from BT-IgG * bound to an avidin-coated plasma treated electrode. 0.5% (w / v) detergent was added to one of the two ECL assay buffers. FIG. 6A shows the results obtained with 150 mM TPA, 250 mM phosphate, pH 7.5. FIG. 6B shows the results obtained using 50 mM PIPES, 150 mM phosphate, pH 7.5. BT-IgG * (50 μL of 0.01 nM solution) was bound to the avidin surface. The plate was washed and ECL assay buffer was added to detect ECL and the plate was analyzed. The figure shows assay buffer background, signal, and S / B ratio (calculated as in Example 4) measured using each of the ECL assay buffers. Of the detergents tested, only Triton X-100 showed a significant effect on performance. In the case of PIPES-based buffers, the effect was significant, with the addition of Triton increasing the S / B ratio> 2.5 times. The effect of Triton X-100 on TPA-based buffer was very slight. Triton X-100 differs from the other cleaning agents used in that it contains a phenol ether component. Applicants hypothesize that the advantageous effects of Triton X-100 are due to the oxidation of the phenol ether component at the electrode and the involvement of Triton oxidation products in the ECL reaction.

  Surprisingly, the effectiveness of detergents on assays performed on non-plasma-treated plates is very different. FIG. 7 shows the effect of five different nonionic detergents on TPA / Phos, TPA / Tris, TPA / Gly-Gly, and PIPES / Phos assay buffers (optimal formulation of Example 4 except for detergent composition and amount). Indicates. The concentrations of Tween 20, Thesit, Triton X-100 and Triton X-114 were all 0.05% (v / v). The concentration of β-octylglucopyranoside was 4% (v / v). In this experiment, streptavidin-coated electrodes were treated with 0.018 pmol BT-IgG * (6.3 labels per protein) in a volume of 50 uL. From the figure it can be seen that for all detergents, the ECL signal measured in the presence of TPA-containing assay buffer is significantly improved over the same assay buffer in the absence of detergent. In most cases, it improved more than twice. In additional experiments, it was observed that the maximum signal observed in each assay buffer tended to occur above the critical micelle concentration (cmc) of the detergent. In contrast to TPA-containing ECL assay buffer, the performance of PIPES was improved approximately 30-fold by the addition of phenol ether-containing detergents (Triton X-100 and Triton X-114), but the presence of Tween and Thesit detergents Only a slight signal improvement was observed below.

  Applicants hypothesize that the effect of Triton detergent on the PIPES / Phos buffer is related to the involvement of Triton oxidation products in the ECL process. In contrast, the effect of the detergent on the ECL signal from the TPA-containing assay buffer on the NPT electrode is a more general phenomenon and is related to the stabilization of the TPA oxidation product in the detergent micelle. looks like.

  A larger screen of detergents was performed to identify other detergents that improve the ECL signal from BT-IgG * on streptavidin-coated NPT electrodes in the presence of TPA / phosphate. All tested non-ionic detergents (APO-14, Triton X-100, β-nonyl glucoside, Tween 20, Genapol and pentaethylene glycol mono-n-dodecyl ether) and zwitterionic detergents (ASB-14 and Empigen) Increased the ECL signal.

ECL activity of selected tertiary amines Several tertiary amines were screened for ability to act as co-reactants. Measurements were carried out in the same manner as described in Examples 4 and 5. An ECL assay buffer containing selected tertiary amine (200 mM), phosphate buffer (200 mM), Triton X-100 (0.1%) and adjusted to pH 7.5 was prepared. The signal from the label attached to the electrode was measured by the following procedure. i) A solution containing 0.3 nM Bt-IgG * (Sulfo-TAG and IgG labeled with biotin) in ECL assay buffer was introduced into the wells of a streptavidin-coated 96-well NPT or PT plate. ii) The plate was incubated for 2 hours with shaking to allow Bt-IgG * to bind to the surface. iii) The plate was washed 4 times and 150 μL of ECL assay buffer was added. iv) ECL from the label was measured. Assay buffer background was measured by introducing 150 uL of ECL assay buffer into streptavidin coated plates and measuring ECL. Free ECL in the solution by introducing a solution containing 10 nM IgG * (IgG labeled with Sulfo-TAG and not labeled with biotin) into the wells of a streptavidin-coated 96-well NPT or PT plate and measuring the ECL The ECL signal from the label was measured.

  Tables IIa and IIb show the experimental results with NPT and PT plates, respectively. The results show that various tertiary amines are suitable for use as co-reactants. In general, it seemed that the ratio of binding signal to free signal was improved by providing functionality. Tertiary amines containing N-substituted morpholine nuclei or even more advantageously di-N-substituted piperazine nuclei (especially PIPES, HEPES, POPSO, HEPPSO and EPPS) can be combined with a binding signal (especially at the NPT surface). It appeared to be particularly suitable for identifying the free signal. There was a slight difference in the relative performance on PT and NPT plates. For example, MOPS has been found to perform particularly well on PT plates, while bis-Tris-propane exhibits an exceptionally high signal on NPT plates.

  ECL was measured using the co-reactant in approximately the same buffer except no detergent or using Tween 20 as the detergent. Two co-reactants other than TPA protruded little depending on the presence or absence of Triton X-100 (N, N-bis- (hydroxyethyl) -N-4-aminobutanesulfonic acid and TPA dimer). These detergents have a binding / release ratio over TPA and are particularly suitable for non-washing assays involving detergent sensitive components.

  Additional experiments have shown that these co-reactants can be used in ECL assay buffers buffered with a variety of different pH buffers, eg, GlyGly, Gly, Tris, Tricine and phosphate.

  The present invention should not be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to the embodiments described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are also intended to be within the scope of the claims. Various publications are cited herein, the entire disclosures of which are incorporated herein by reference.

FIG. 6 is a graph comparing the dissociation rates of phosphopeptide-anti-phosphopeptide complexes in three different ECL assay buffers containing different pH buffers. FIG. 6 is a graph showing the dissociation rate of a phosphopeptide-antiphosphopeptide complex in an ECL assay buffer containing TPA as an ECL co-reactant and Tris as a pH buffer. The complex was not washed to remove free antibody prior to addition of ECL assay buffer. Of the final product comparing the dissociation rate of anti-phosphotyrosine antibody from autophosphorylated EGF receptor in two different ECL assay buffers: 150 mM TPA / 150 mM phosphate and 100 mM TPA / 400 mM glycyl-glycine It is a graph which shows a stability test. The concentration of labeled α-phosphotyrosine antibody was 6.7 nM. FIG. 6 is a graph comparing the performance of four different ECL assay buffers in the ECL measurement of a labeling reagent immobilized on the surface of an unetched carbon ink electrode. This graph shows the signal from the reagent bound to the surface, the background signal in the absence of binding reagent, and the signal / background ratio (S / B). FIG. 6 is a graph comparing the performance of four different ECL assay buffers in an ECL measurement of a labeling reagent immobilized on the surface of a plasma etched carbon ink electrode. This graph shows the signal from the reagent bound to the surface, the background signal measured in the absence of binding reagent, and the signal / background ratio (S / B). FIG. 6 is a graph comparing the performance of four different ECL assay buffers in the ECL measurement of a labeled reagent immobilized on an unetched carbon ink electrode surface. This graph shows the signal from the reagent bound to the surface, the background signal measured in the absence of binding reagent and the signal / background ratio (S / B). This figure also shows the signal obtained when a labeling reagent that is not bound to the surface is introduced into the ECL assay buffer, and the signal ratio (B / F) between surface bound and surface unbound reagent. FIG. 6 is a graph comparing the performance of four different ECL assay buffers in the ECL measurement of a labeling reagent immobilized on a plasma etched carbon ink electrode surface. This graph shows the signal from the reagent bound to the surface, the background signal measured in the absence of binding reagent and the signal / background ratio (S / B). This figure also shows the signal obtained when a labeling reagent that is not bound to the surface is introduced into the ECL assay buffer, and the signal ratio (B / F) between surface bound and surface unbound reagent. FIG. 5 is a graph comparing the effect of three different cleaning agents on the ECL signal from a labeling reagent immobilized on a plasma etched carbon ink electrode surface. The detergent was introduced into an ECL assay buffer containing TPA and phosphate. This graph shows the signal from the reagent bound to the surface, the background signal measured in the absence of binding reagent, and the signal / background ratio (S / B). FIG. 5 is a graph comparing the effect of three different cleaning agents on the ECL signal from a labeling reagent immobilized on a plasma etched carbon ink electrode surface. Detergent was introduced into ECL assay buffer containing PIPES and phosphate. This graph shows the signal from the reagent bound to the surface, the background signal measured in the absence of binding reagent, and the signal / background ratio (S / B). FIG. 6 is a graph comparing the effect of five different detergents on the ECL signal from a labeling reagent immobilized on an unetched carbon ink electrode surface. Detergents were introduced into four different ECL assay buffers with different types of ECL co-reactants or pH buffers. This figure shows the signal from the reagent bound to the surface, the background signal measured in the absence of binding reagent, and the signal / background ratio (S / B).

Claims (169)

  A composition comprising a pH buffer and a phosphate-specific antibody and substantially free of inorganic phosphate.   A composition suitable for an assay comprising a pH buffer and a phosphate-specific antibody and substantially free of inorganic phosphate.   A composition comprising a pH buffer, a phosphopeptide-specific antibody, and a phosphopeptide that binds to the phosphopeptide-specific antibody and substantially free of inorganic phosphate.   4. A composition according to claim 1, 2 or 3, wherein the pH buffer is selected from the group consisting of glycylglycine, tris (hydroxymethyl) aminomethane or combinations thereof.   5. The composition of claim 1, 2, 3 or 4 further comprising one or more kinases and / or one or more kinase substrates.   5. The composition of claim 1, 2, 3 or 4 further comprising one or more phosphatases and / or one or more phosphatase substrates.   The composition according to claim 1, 2, 3, 5 or 6, wherein the pH buffer is glycylglycine.   The composition of claim 1, 2, 3, 5 or 6, wherein the pH buffer is tris [hydroxymethyl] aminomethane.   A composition according to any preceding claim, wherein the composition comprises one or more ECL coreactants.   The composition of claim 9, wherein the ECL co-reactant comprises a tertiary amine.   The composition of claim 9, wherein the ECL coreactant comprises tripropylamine (TPA).   A composition according to any preceding claim, further comprising KOH.   The composition according to any of the preceding claims, further comprising a cleaning agent or a surfactant.   The composition according to any of the preceding claims, further comprising a non-ionic detergent or surfactant.   15. The composition of any one of claims 1, 2, or 4-14, further comprising a phosphopeptide that binds to the phosphopeptide-specific antibody.   A composition according to any preceding claim, wherein the composition does not contain inorganic phosphate.   A composition according to any preceding claim, further comprising a stop reagent.   The composition according to any of the preceding claims, further comprising ethylenediaminetetraacetic acid (EDTA).   The composition according to any of the preceding claims, further comprising a base.   A composition according to any preceding claim, further comprising KOH.   The composition according to any one of claims 1 to 18, further comprising KOH and a surfactant or a cleaning agent.   A composition according to any preceding claim, further comprising an electrochemiluminescent label.   A composition according to any preceding claim, further comprising a metal-containing electrochemiluminescent label.   A composition according to any preceding claim, further comprising a Ru or Os containing electrochemiluminescent label.   The composition according to any of the preceding claims, wherein the pH of the composition is 6-9.   The composition according to any of the preceding claims, wherein the pH of the composition is 7-8.   The composition according to any of the preceding claims, wherein the pH of the composition is 7.5-8.   A composition according to any preceding claim, wherein the pH of the composition is about 7.8.   30. A device configured to perform an electrochemiluminescence assay comprising a composition according to any one of claims 1 to 28.   30. The apparatus of claim 29, further comprising a photodetector that detects light emitted from the composition and / or a voltage source that applies a voltage to the composition sufficient to induce electrochemiluminescence.   29. A kit comprising an assay module comprising a composition according to any one of claims 1 to 28 and one or more working electrodes in one or more containers.   A method for performing an electrochemiluminescence method comprising applying electrochemical energy to a composition according to any one of claims 1 to 28.   35. The method of claim 32, further comprising detecting the emitted electrochemiluminescence. A method of performing an assay using a composition according to any one of claims 1-28, comprising:
Forming an assay mixture comprising the composition and a kinase product; and forming a complex comprising the kinase product and the phosphate-specific antibody.   35. The method of claim 34, further comprising contacting a kinase with a kinase substrate to form the kinase product and detecting the complex.   36. A method according to claim 34 or 35, wherein the complex is detected using electrochemiluminescence.   A method of performing an assay comprising forming a complex comprising a kinase product and a phosphate-specific antibody and not exposing the complex to inorganic phosphate.   Contacting the kinase with a kinase substrate to form the kinase product; contacting the kinase product with the phosphate-specific antibody to form the complex; and detecting the complex. 38. The method of claim 37 comprising.   39. The method of claim 37 or 38, wherein the complex is detected using electrochemiluminescence. A composition suitable for use in an electrochemiluminescence method comprising a pH buffer and an ECL co-reactant comprising:
The pH buffer is selected from the group consisting of glycylglycine, tris [hydroxymethyl] aminomethane, or combinations thereof, and the ECL co-reactant comprises a tertiary amine for use in an electrochemiluminescence method. Suitable composition.   41. The composition of claim 40, wherein the ECL coreactant is tripropylamine.   A composition suitable for use in an electrochemiluminescence method comprising a pH buffer that is not an inorganic phosphate, a Ru or Os containing ECL label, and an ECL co-reactant.   43. The composition of claim 42, wherein the ECL coreactant comprises a tertiary amine.   44. The composition of claim 43, wherein the ECL coreactant is tripropylamine.   A composition suitable for use in an electrochemiluminescence method comprising a pH buffer that is not an inorganic phosphate and an ECL co-reactant that is tripropylamine.   46. The composition of claim 42, 43, 44 or 45, wherein the pH buffer is an ECL assay pH buffer background reducing agent.   47. The composition of claim 40, 41, 42, 43, 44, 45 or 46, wherein the concentration of the co-reactant is 50 to 1000 mM.   A composition suitable for use in an electrochemiluminescence method, comprising (a) tripropylamine having a concentration in the range of 50-1000 mM, and (b) having a concentration in the range of 50-1000 mM, and glycylglycine , Tris [hydroxymethyl] -aminomethane, or a pH buffer selected from the group consisting of combinations thereof, the composition having a pH in the range of 6.5 to 8.5.   49. The composition of claim 40, 41, 45 or 48, further comprising an ECL component.   50. The composition of claim 49, wherein the ECL component is a metal-containing ECL component.   51. The composition of claim 50, wherein the ECL component is a Ru or Os containing ECL label.   52. The composition of claim 42, 43, 44, 49, 50 or 51, wherein the ECL component is capable of repeatedly releasing electrochemiluminescence.   53. A composition according to any of claims 40 to 52, wherein the concentration of inorganic phosphate is less than 20 mM.   53. A composition according to any of claims 40 to 52, wherein the composition is substantially free of inorganic phosphate.   53. A composition according to any of claims 40 to 52, wherein the composition does not comprise inorganic phosphate.   56. The composition of claim 54 or 55, further comprising one or more components selected from the group consisting of a kinase and a kinase substrate.   56. The composition of claim 54 or 55, further comprising one or more components selected from the group consisting of phosphatases and phosphatase substrates.   58. A composition according to any one of claims 40 to 57, wherein the pH buffering agent is glycylglycine.   58. The composition according to any one of claims 40 to 57, wherein the pH buffer is tris [hydroxymethyl] aminomethane.   60. The composition according to any one of claims 40 to 59, further comprising ethylenediaminetetraacetic acid (EDTA).   61. The composition according to any one of claims 40 to 60, further comprising KOH.   62. A composition according to any one of claims 40 to 61, further comprising a substance selected from the group consisting of salts, detergents, preservatives, chelating agents and antifoaming agents.   63. The composition according to any one of claims 40 to 62, further comprising a surfactant.   64. The composition according to any one of claims 40 to 63, wherein the pH of the composition is 6.5 to 8.5.   The composition according to any one of claims 40 to 64, wherein the pH of the composition is 7-8.   66. A composition according to any one of claims 40 to 65, wherein the pH of the composition is 7.5-8.   67. A composition according to any one of claims 40 to 66, wherein the pH of the composition is about 7.8.   68. A composition according to any one of claims 40 to 67, wherein the composition is aqueous.   69. A composition according to any one of claims 40 to 68, wherein the composition provides a suitable environment for deriving ECL from a metal-containing ECL component under oxidizing conditions.   70. A reagent suitable for use in an electrochemiluminescence method comprising the composition according to any one of claims 40 to 69. A reagent comprising a pH buffer substantially free of inorganic phosphate and a Ru or Os containing ECL component,
The reagent is suitable for use in a composition for performing an ECL assay,
(I) a metal-containing ECL component that is convertible to an excited state from which electromagnetic radiation is emitted;
(Ii) an ECL coreactant that forms a strong reducing agent when oxidized;
(Iii) an electrolyte capable of functioning as a medium in which the ECL component and the amine or amine component can be oxidized;
A reagent wherein electromagnetic radiation is emitted by an assay composition comprising a component selected from the group consisting of:   72. The reagent of claim 71, wherein the reagent further comprises the pH buffer, an amine or an amine component, and one of the other two components of the groups (i)-(iii).   72. The reagent of claim 71, further comprising a surfactant or detergent.   72. The reagent of claim 71, further comprising an ECL co-reactant.   72. The reagent of claim 71, further comprising EDTA.   In one or more containers, comprising a pH buffer and an ECL co-reactant, wherein the pH buffer is selected from the group consisting of glycylglycine, tris [hydroxymethyl] aminomethane, or combinations thereof, and the ECL co-reactant. A kit suitable for use in an electrochemiluminescent method wherein the reactant comprises a tertiary amine.   In an electrochemiluminescent method, wherein the one or more containers comprise a pH buffer, a Ru or Os containing ECL label, and an ECL co-reactant, wherein the composition is substantially free of inorganic phosphate. A kit suitable for use.   A kit suitable for use in an electrochemiluminescence method comprising a pH buffer and tripropylamine in one or more containers, wherein the composition is substantially free of inorganic phosphate.   In one or more containers, a concentration 50 selected from the group consisting of (a) tripropylamine at a concentration of 50-1000 mM and (b) glycylglycine, tris [hydroxymethyl] -aminomethane, or combinations thereof. A kit suitable for use in an electrochemiluminescence method comprising ˜1000 mM pH buffer.   79. The kit of claim 78, comprising tripropylamine at a concentration of 50-1000 mM.   The kit according to claim 76, 77, 78, 79 or 80, which does not contain inorganic phosphate.   82. The kit of claim 76, 77, 78, 79, 80 or 81, further comprising one or more components selected from the group consisting of a kinase and a kinase substrate.   82. The kit of claim 76, 77, 78, 79, 80 or 81, further comprising one or more components selected from the group consisting of phosphatases and phosphatase substrates.   84. The kit according to any one of claims 76 to 83, wherein the pH buffer is glycylglycine.   84. The kit according to any one of claims 76 to 83, wherein the pH buffer is tris [hydroxymethyl] aminomethane.   84. A kit according to any one of claims 77 or 81 to 83, wherein the ECL co-reactant comprises a tertiary amine.   78. The kit of claim 76 or 77, wherein the ECL coreactant comprises tripropylamine (TPA).   The kit according to any one of claims 76 to 87, further comprising ethylenediaminetetraacetic acid (EDTA).   79. The kit of claim 76, 77 or 78, wherein the pH buffer is glycylglycine and the composition further comprises ethylenediaminetetraacetic acid (EDTA).   90. The kit according to any one of claims 76 to 89, further comprising KOH.   The kit according to any one of claims 76 or 78 to 90, further comprising an ECL component.   The kit according to any one of claims 76 or 78 to 90, further comprising an ECL component capable of repeatedly releasing electrochemiluminescence.   The kit according to any one of claims 76 or 78 to 90, further comprising a metal-containing ECL component.   The kit according to any one of claims 76 or 78 to 90, further comprising an ECL label containing Ru or Os.   95. The kit according to any one of claims 76 to 94, further comprising one or more substances selected from the group consisting of salts, cleaning agents, preservatives, chelating agents and antifoaming agents.   96. The kit according to any one of claims 76 to 95, further comprising a surfactant.   97. A kit according to any one of claims 76 to 96, wherein the kit is aqueous.   98. Any one of claims 76, 78-89 or 95-97, further comprising at least one separate component including one or more components of the group consisting of ECL component (i) and electrolyte (ii). Kit according to item.   A method of performing a specific binding assay qualitatively or quantitatively in a composition comprising (a) a pH buffer substantially free of inorganic phosphate and (b) a phosphate-specific antibody.   100. The method of claim 99, wherein the composition further comprises an ECL co-reactant.   100. The method of claim 99, wherein the composition further comprises an ECL label.   102. The method of claim 101, wherein the composition further comprises a phosphate specific antibody.   Qualitatively or quantitatively specific using a composition comprising a pH buffer substantially free of inorganic phosphate in a well comprising one or more assay domains immobilized thereon. A method of performing a bound unwashed assay. An electrochemiluminescent label;
At least one reagent selected from the group consisting of a kinase, a kinase substrate, a kinase product, and combinations thereof;
A method of performing a kinase assay using a composition comprising: a composition substantially free of inorganic phosphate.   105. The method of claim 104, further comprising providing electrochemical energy effective to induce an electrochemiluminescent label to emit electromagnetic radiation and detecting the emitted electrochemiluminescence.   106. The method of claim 104 or 105, further comprising contacting the composition with a phosphate specific antibody. A method for performing an assay comprising:
(A) forming a complex comprising a kinase product and a phosphate-specific antibody, and not exposing the complex to inorganic phosphate;
(B) inducing a metal-containing ECL component to emit electromagnetic radiation;
(C) detecting the emitted electromagnetic radiation.   108. The method of claim 107, wherein the complex further comprises the metal-containing ECL component.   A method of inducing electrochemiluminescence induced in the presence of a composition comprising an ECL label, an ECL co-reactant, and a pH buffer that is not phosphate.   110. The method of claim 109, wherein the pH buffer is an ECL assay pH buffer background reducing agent.   110. The method of claim 109, wherein the pH buffer is selected from the group consisting of glycylglycine, tris [hydroxymethyl] aminomethane, or combinations thereof.   112. The method of claim 109, 110 or 111, wherein the composition is substantially free of inorganic phosphate.   113. The method of claim 109, 110, 111 or 112, wherein the ECL co-reactant is a tertiary amine.   The co-reactant is present at a concentration of 50-1000 mM, the pH buffer is present at a concentration of 50-1000 mM, the pH is 6.5-8.5, and the ECL co-reactant is tripropylamine. 110. The method of claim 109, wherein the pH buffering agent is selected from the group consisting of glycylglycine, tris [hydroxymethyl] -aminomethane, or combinations thereof.   115. The method according to any one of claims 109 to 114, wherein the ECL label is a ruthenium or osmium organometallic complex. The method further comprises:
(A) exposing the composition to electrochemical energy to induce the electrochemiluminescent composition to emit electromagnetic radiation;
116. A method according to any one of claims 109 to 115, comprising (b) detecting emitted electromagnetic radiation.   117. The method of claim 116, wherein the electrochemical energy is generated by applying an oxidation potential to an electrode.   118. The method of claim 117, wherein the ECL label is attached to the electrode.   119. A method according to claim 117 or 118, wherein the electrode comprises elemental carbon.   An electrochemiluminescent assay pH buffer comprising an ECL coreactant that is a tertiary amine and at least one non-phosphate based pH buffer.   121. The electrochemiluminescent assay pH buffer of claim 120, wherein the co-reactant is TPA.   121. The electrochemiluminescent assay buffer of claim 120, wherein the non-phosphate based pH buffer is selected from tris [hydroxymethyl] aminomethane (Tris), oligo (glycine), or combinations thereof.   121. The electrochemiluminescent assay buffer of claim 120, wherein the non-phosphate based pH buffer is glycyl-glycine (Gly-Gly).   124. The electrochemiluminescence assay according to any one of claims 120 to 123, further comprising one or more substances selected from the group consisting of chelating agents, cleaning agents, salts, antifoaming agents and preservatives. Buffer.   125. The electrochemiluminescence assay buffer according to any one of claims 120 to 124, further comprising a phosphate specific antibody.   125. Contacting an assay buffer according to any one of claims 120 to 125 with an ECL label, inducing electrochemiluminescence, and detecting the released electrochemiluminescence. A method of generating chemiluminescence.   A composition comprising an electrochemiluminescent label, a non-TPA electrochemiluminescent co-reactant, and at least one phenyl ether-containing detergent.   128. The composition of claim 127, wherein the non-TPA electrochemiluminescent co-reactant is PIPES.   The non-TPA electrochemiluminescent co-reactant is 3- (di-n-propylamino) -propanesulfonic acid, 4- (di-n-propylamino) -butanesulfonic acid, 4- [bis- (2-hydroxy Ethane) -amino] -butanesulfonic acid, piperidine-N- (3-propanesulfonic acid), azepane-N- (3-propanesulfonic acid), piperidine-N- (3-propionic acid) (PPA), 3- Morpholino-2-hydroxypropanesulfonic acid (MOPSO), 3-morpholinepropanesulfonic acid (MOPS), N- (2-hydroxyethyl) piperazine-N′-3-propanesulfonic acid (EPPS), N- (2-hydroxy Ethyl) piperazine-N′-3-ethanesulfonic acid (BES), piperazine-N, N′-bis (2-ethanesulfonic acid) (PI) ES), triethanolamine, N-2-hydroxypiperazine-N-2-ethanesulfonic acid (HEPES), piperazine-N, N′-bis-4-butanesulfonic acid, homopiperidine-N-3-propanesulfonic acid Piperazine-N, N′-bis-3-propanesulfonic acid, piperidine-N-3-propanesulfonic acid, piperazine-N-2-hydroxyethane-N′-3-methylpropanoate, piperazine-N, N '-Bis-3-methylpropanoate, 1,6-diaminohexane-N, N, N', N'-tetraacetic acid, N, N-bispropyl-N-4-aminobutanesulfonic acid, N- Tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (TES), 1,3-bis [tris (hydroxymethyl) methylamino] propane (bis- rispropane), 3-dimethylamino-1-propanol, 3-dimethylamino-2-propanol, N, N, N ′, N′-tetrapropylpropane-1,3-diamine (TPA dimer), piperazine- Consists of N, N′-bis (2-hydroxypropane) sulfonic acid (POPSO) and 2-hydroxy-3- [4- (2-hydroxyethyl) piperazin-1-yl] propane-1-sulfonic acid (HEPPSO) 128. The composition of claim 127, selected from the group.   130. The composition of claim 127, 128 or 129, wherein the composition is in contact with a working electrode.   130. The composition of claim 127, 128 or 129, wherein the composition is in contact with a carbon-based working electrode.   132. A composition according to any of claims 127 to 131, wherein the electrochemiluminescent label is a Ru or Os containing ECL label.   A composition comprising 3- (di-n-propylamino) -propanesulfonic acid or 4- (di-n-propylamino) -butanesulfonic acid.   134. The composition of claim 133, further comprising an ECL label.   134. The composition of claim 133, further comprising a metal-containing ECL label.   134. The composition of claim 133, further comprising a Ru or Os containing ECL label.   A method of inducing electrochemiluminescence using the composition according to any one of claims 133 to 136. A method for generating electrochemiluminescence, comprising:
Contacting the electrochemiluminescent label with the composition of claim 136;
(B) inducing the electrochemiluminescent label to emit electrochemiluminescence;
(C) detecting the electrochemiluminescence. A method for generating electrochemiluminescence, comprising:
(A) inducing a composition according to any one of claims 133 to 137 to emit electrochemiluminescence;
(B) detecting the electrochemiluminescence.   140. The method of claim 138 or 139, wherein the electrochemiluminescent label is attached to a working electrode.   140. The method of claim 138 or 139, wherein the ECL induction is performed in a composition that is substantially free of detergent.   A kit comprising, in one or more containers, an electrochemiluminescent label, a non-TPA electrochemiluminescent co-reactant, and at least one phenyl ether-containing detergent.   A kit comprising one or more ECL assay buffers comprising at least one trialkylamine non-TPA co-reactant in one or more containers.   In one or more containers, a metal-containing electrochemiluminescent label and 3- (di-n-propylamino) -propanesulfonic acid, 4- (di-n-propylamino) -butanesulfonic acid and combinations thereof A kit comprising at least one co-reactant selected from the group consisting of: A method for generating electrochemiluminescence, comprising:
(A) contacting an electrochemiluminescent label with an electrochemiluminescent co-reactant;
(B) inducing the electrochemiluminescent label to emit electrochemiluminescence;
(C) detecting the electrochemiluminescence,
The method wherein the electrochemiluminescent co-reactant is a tertiary amine other than TPA. A method of inducing electrochemiluminescence comprising contacting an electrochemiluminescent label with an electrochemiluminescent co-reactant comprising:
A method for inducing electrochemiluminescence, wherein the electrochemiluminescent co-reactant is a tertiary amine other than tri-n-propylamine, and the electrochemiluminescent label is attached to a working electrode. A method for performing an electrochemiluminescent assay comprising:
(A) forming a composition comprising an electrochemiluminescent label and at least one electrochemiluminescent co-reactant, wherein the electrochemiluminescent co-reactant is a third other than tri-n-propylamine. A step, wherein the electrochemiluminescent label is attached to the working electrode;
(B) providing electrochemical energy effective to induce the electrochemiluminescent label to emit electrochemiluminescence;
(C) detecting the emitted electrochemiluminescence. A method for performing an electrochemiluminescent assay comprising:
(A) forming a composition comprising an electrochemiluminescent label and at least one electrochemiluminescent co-reactant, wherein the electrochemiluminescent co-reactant is a tertiary other than tri-n-propylamine An amine, wherein the first part of the electrochemiluminescent label is attached to the working electrode, and the second part of the electrochemiluminescent label is in solution;
(B) providing electrochemical energy effective to induce a first portion of the electrochemiluminescent label to emit electrochemiluminescence;
(C) selectively detecting electrochemiluminescence emitted from the first portion of the electrochemiluminescent label. A method of performing an assay of interest analyte or activity comprising:
(A) forming a composition comprising a sample, an assay reagent comprising an ECL label, and an electrode, wherein the presence of the analyte or activity in the sample results in the assay reagent from the electrode Steps resulting in attachment or dissociation;
(B) contacting the electrode with an ECL assay buffer containing an ECL co-reactant, wherein the co-reactant is a tertiary amine other than TPA;
(C) inducing ECL labeling on the electrode to provide electrochemical energy to the electrode under conditions suitable to induce ECL;
(D) measuring the released ECL. A method for measuring a binding phenomenon,
(A) contacting a binding reagent immobilized on an electrode with an assay reagent comprising an ECL label, wherein the binding reagent specifically binds to the assay reagent;
(B) binding the assay reagent to the binding reagent and attaching the label to the electrode;
(C) contacting the electrode with an ECL assay buffer comprising an ECL co-reactant, wherein the co-reactant is a tertiary amine other than TPA;
(D) inducing ECL labeling on the electrode to provide electrochemical energy to the electrode under conditions suitable to induce ECL;
(E) measuring the released ECL. The co-reactant has one or more of the following properties: i) is oxidized on a carbon-based electrode in a one-electrode oxidation to give an amine radical cation, and the amine radical cation subsequently loses a proton. A radical reducing agent can be formed, and ii) has an oxidation potential equal to or greater than that of Ru (II) (bpy) _{3 on} the carbon base electrode, and iii) decomposes water at the carbon base electrode And iv) the energy released by the reaction of the radical reducing agent and Ru (III) (bpy) ₃ reacting to form Ru (II) (bpy) _3. Sufficient to produce Ru (II) (bpy) ₃ in the luminescent excited state, and v) the lifetime of the amine radical cation and / or radical reducing agent is 150. The method according to any of claims 145 to 150, wherein the method is shorter than a TPA-derived species that   152. The co-reactant forms an amine radical cation and / or a radical reducing agent, and the diffusion distance of the amine radical cation and / or radical reducing agent is less than 100 nm, according to any one of claims 145 to 151. the method of.   153. A method according to any one of claims 145 to 152, wherein the method is performed without a washing step.   154. Replacing the ECL co-reactant with TPA reduces the selectivity of ECL induction from a label attached to the electrode relative to the label in solution by at least a factor of two. The method described in 1.   157. A method according to any of claims 145 to 154, wherein the electrochemiluminescent co-reactant is PIPES. The co-reactant is an amine having a structure of NR ¹ R ² R ³ , wherein R ¹ , R ² and R ³ are alkyl groups containing at least two carbons, and R ¹ , R ² and R 3 one or more of the ^three has been functionalized with hydrophilic functional groups, the method according to any one of claims 145 to 154.   157. The method of claim 156, wherein the hydrophilic functional group comprises a charged group.   158. The method of claim 157, wherein the hydrophilic functional group comprises a negatively charged group.   158. The method of claim 157, wherein the hydrophilic functional group comprises hydroxyl, dialkylamino, sulfate, sulfonate, carboxylate and / or carboxylic acid ester. The coreactant has a _{(n-Pr) 2 N (} CH 2) n structure R, wherein, n is 2 or more, R is hydrophilic functional groups, of claim 145 to 154 The method according to any one of the above. The co-reactant has the following formula: i) X is — (CH ₂ ) — or a heteroatom, ii) R and R ¹ are alkyl groups containing two or more carbons, and iii) n 155. The method of any one of claims 145 to 154, wherein and m are each 1 or more, and iv) R (and optionally R ¹ ) comprises a hydrophilic functional group.

  The co-reactant is 3- (di-n-propylamino) -propanesulfonic acid, 4- (di-n-propylamino) -butanesulfonic acid, 4- [bis- (2-hydroxyethane) -amino]. -Butanesulfonic acid, piperidine-N- (3-propanesulfonic acid), azepane-N- (3-propanesulfonic acid), piperidine-N- (3-propionic acid) (PPA), 3-morpholino-2-hydroxy Propanesulfonic acid (MOPSO), 3-morpholinepropanesulfonic acid (MOPS), N- (2-hydroxyethyl) piperazine-N′-3-propanesulfonic acid (EPPS), N- (2-hydroxyethyl) piperazine-N '-3-ethanesulfonic acid (BES), piperazine-N, N'-bis (2-ethanesulfonic acid) (PIPES), triethane Ruamine, N-2-hydroxypiperazine-N-2-ethanesulfonic acid (HEPES), piperazine-N, N′-bis-4-butanesulfonic acid, homopiperidine-N-3-propanesulfonic acid, piperazine-N, N′-bis-3-propanesulfonic acid, piperidine-N-3-propanesulfonic acid, piperazine-N-2-hydroxyethane-N′-3-methylpropanoate, piperazine-N, N′-bis-3 -Methylpropanoate, 1,6-diaminohexane-N, N, N ', N'-tetraacetic acid, N, N-bispropyl-N-4-aminobutanesulfonic acid, N-tris (hydroxymethyl) Methyl-2-aminoethanesulfonic acid (TES), 1,3-bis [tris (hydroxymethyl) methylamino] propane (bis-Trispropane), -Dimethylamino-1-propanol, 3-dimethylamino-2-propanol, N, N, N ', N'-tetrapropylpropane-1,3, -diamine (TPA dimer), piperazine-N, N'- Selected from the group consisting of bis (2-hydroxypropane) sulfonic acid (POPSO) and 2-hydroxy-3- [4- (2-hydroxyethyl) piperazin-1-yl] propane-1-sulfonic acid (HEPPSO) 155. A method according to any one of claims 145 to 154.   157. Any one of claims 145 to 154, wherein the co-reactant is selected from the group consisting of piperidine-N- (3-propionic acid) and piperazine-N, N′-bis (2-ethanesulfonic acid). The method described in 1.   164. The method according to any one of claims 145 to 163, wherein the electrochemiluminescence is induced using one or more carbon-based working electrodes.   166. The method of claim 164, wherein the one or more carbon-based working electrodes comprise carbon particles or carbon nanotubes.   166. A method according to any one of claims 145 to 165, wherein the ECL label is also contacted with at least one phenyl ether containing detergent.   173. The method according to any one of claims 145 to 166, wherein the concentration of the co-reactant is 10 to 800 mM.   168. The method of any one of claims 146 to 167, further comprising contacting the label with a pH buffer.   168. The method of claim 168, wherein the concentration of the pH buffer is preferably 0-800 mM.

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